Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells

ABSTRACT

The invention provides a novel retroviral packaging system, in which retroviral packaging plasmids and packagable vector transcripts are produced from high expression plasmids after stable or transient transfection in mammalian cells. High titers of recombinant retrovirus are produced in these transfected mammalian cells and can then transduce a mammalian target cell by cocultivation or supernatant infection. The methods of the invention include the use of the novel retroviral packaging plasmids and vectors to transduce primary human cells, including T cells and human hematopoietic stem cells, with foreign genes by cocultivation or supernatant infection at high efficiencies. The invention is useful for the rapid production of high titer viral supernatants, and to transduce with high efficiency cells that are refractory to transduction by conventional means.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/944,411,filed Sep. 4, 2001, now U.S. Pat. No. 6,506,604, which is a continuationof application Ser. No. 08/914,893, filed Aug. 20, 1997, abandoned,which is a continuation of application Ser. No. 08/517,488. filed Aug.21, 1995, now U.S. Pat. No. 6,051,427 which is a continuation-in-part ofapplication Ser. No. 08/258,152 filed Jun. 10, 1994, now U.S. Pat. No.5,686,279, which is a continuation-in-part of application Ser. No.08/076,299, filed Jun. 11, 1993, now U.S. Pat. No. 5,834,256, thedisclosures of both the 08/258,152 and 08/076,299 applications areincorporated by reference in their entirety herein.

FIELD OF THE INVENTION

This invention relates to novel retrovirus packaging plasmids andvectors, to their use in the production of recombinant retrovirus inmammalian cells, and to methods of using such constructs to transducemammalian target cells with high efficiency. The invention also relatesto the construction of stable cell lines in which novel retroviralpackaging plasmids and/or vectors are stably expressed in viralpackaging cell lines.

BACKGROUND OF THE INVENTION

Retrovirus vectors have become the primary tool for gene delivery inhuman gene therapy applications (Miller, Nature 357:455-460 (1992)). Theability of retrovirus vectors to deliver an unrearranged, single copygene into a broad range of rodent, primate and human somatic cells inprimary culture makes them well suited for this purpose. Identificationand subsequent deletion of the sequences present within retroviraltranscripts encoding the packaging signals for avian (E) and murine (ψ)retroviruses, has enabled development of packaging cell lines to supplyin trans the proteins necessary for production of infectious virions,but render the packaging cell lines unable to package their own viralgenomic mRNA (Watanabe and Temin, Molec. Cell. Biol. 3 (12): 2241-2249(1983); Mann et al., Cell 33:153-159 (1983); and Embretson and Temin, J.Virol. 61 (9): 2675-2683 (1987)). The most important consideration inthe construction of retroviral packaging lines has been both theproduction of high titer vector supernatants free of recombinantreplication competent retrovirus, which has been shown to produce T celllymphomas in rodents (Cloyd et al., J. Exp. Med. 151,542-552 (1980)) andprimates (Donahue et al., J. Exp. Med. 176,1125-1135 (1992)). Althoughearly murine retroviral packaging lines were highly prone to generationof replication competent retrovirus (RCR) (Cone and Mulligan, Proc.Nat'l. Acad. Sci. USA 81:6349-6353 (1984)) or prone to co-package theψ-genome (Mann et al., supra, 1983; Buttimore and Miller, Mol. Cell.Biol. 6 (8): 2895-2902 (1986)), two strategies have evolved for theconstruction of second generation packaging lines with significantlyreduced ability for the generation of RCR. One strategy, embodied byPA317, uses a single genome packaging construct from which theinitiation site for second strand synthesis, the 3′ LTR, and the ψ sitehave been deleted (Miller and Buttimore, Molec. Cell. Biol. 6 (8):2895-2902 (1986)). These modifications eliminate as much as possiblehomology between the packaging genome and the viral vector to reduce theability to form recombinants, and have resulted in production of hightiter, helper-free virus with many vector systems (Miller and Rosman,BioTechniques 7 (9): 980-990 (1989)). The second approach has been todivide the packaging functions into two genomes: one that expresses thegag and pol gene products, and the other that expresses the env geneproduct (Bosselman et al., Molec. Cell. Biol. 7 (5): 1797-1806 (1987);Markowitz et al., J. Virol. 62 (4): 1120-1124 (1988); Danos andMulligan, Proc. Nat'l. Acad. Sci. (USA) 85:6460-6464 (1988)). Thisapproach eliminated the ability for co-packaging and subsequent transferof the ψ-genome, as well as significantly decreased the frequency ofrecombination due to the presence of three retroviral genomes in thepackaging cell that must undergo recombination to produce RCR. In theevent recombinants arise, mutations (Danos and Mulligan, supra) ordeletions (Boselman et al., supra; and Markowitz et al., supra) withinthe undesired gene products render recombinants non-functional. Inaddition, deletion of the 3′ LTR on both packaging function constructsfurther reduces the ability to form functional recombinants. Althoughearly attempts at the generation of two genome packaging lines yieldedlow titer producer clones (Bosselman et al., supra) producer lines arenow available that yield high titer producer clones (Danos and Mulligan,supra; and Markowitz et al., supra).

Packaging lines currently available yield producer clones of sufficienttiter to transduce human cells for gene therapy applications and haveled to the initiation of human clinical trials (Miller, supra). However,there are two areas in which these lines are deficient. First, design ofthe appropriate retroviral vectors for particular applications requiresthe construction and testing of several vector configurations. Forexample, Belmont et al., Molec. and Cell. Biol. 8 (12): 5116-5125(1988), constructed stable producer lines from 16 retroviral vectors inorder to identify the vector capable of producing both the highest titerproducer and giving optimal expression. Some of the configurationsexamined included: (1) LTR driven expression vs. an internal promoter;(2) selection of an internal promoter derived from a viral or a cellulargene; and (3) whether a selectable marker was incorporated in theconstruct. A packaging system that would enable rapid, high-titer virusproduction without the need to generate stable producer lines would behighly advantageous in that it would save approximately two monthsrequired for the identification of high titer producer clones derivedfrom several constructs.

Second, compared to NIH 3T3 cells, the infection efficiency of primarycultures of mammalian somatic cells with a high titer amphotropicretrovirus producer varies considerably. The transduction efficiency ofmouse myoblasts (Dhawan et al., Science 254:1509-1512 (1991) or ratcapillary endothelial cells (Yao et. al., Proc. Natl. Acad. Sci. USA88:8101-8105 (1991)) was shown to be approximately equal to that of NIH3T3 cells, whereas the transduction efficiency of canine hepatocytes(Armentano et. al., Proc. Natl. Acad. Sci. USA 87:6141-6145 (1990)) wasonly 25% of that found in NIH 3T3 cells. Primary humantumor-infiltrating lymphocytes (“TILs”), human CD4+ and CD8+ T cellsisolated from peripheral blood lymphocytes, and primate long-termreconstituting hematopoietic stem cells, represent an extreme example oflow transduction efficiency compared to NIH 3T3 cells. Purified humanCD4+ and CD8+ T Cells have been reported on one occasion to be infectedto levels of 6%-9% with supernatants from stable producer clones(Morecki et al., Cancer Immunol. Immunother. 32:342-352 (1991)), andprimate or human long-term reconstituting hematopoietic stem cells haveonly been infected to ≦1% with a producer of titer of 10⁶ per ml on NIH3T3 cells (van Beusechem et al., Proc. Natl. Acad. Sci. USA 89:7640-7644(1992); and Donahue et al. ,supra). If the retrovirus vector containsthe neo^(R) gene, populations that are highly enriched for transducedcells can be obtained by selection in G418. However, selectable markerexpression has been shown to have deleterious effects on long-term geneexpression in vivo in hematopoietic stem cells (Apperly et. al. Blood78:310-317 (1991)).

An approach that yields significantly increased transduction ofmammalian cells in primary culture would be highly advantageous, andthis need is currently unmet.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides novel plasmid basedexpression vectors that direct the synthesis of both packagableretroviral vector transcripts and retroviral gene products required forrapid production of high titer recombinant retrovirus in human cells bytransient transfection, thereby eliminating the need to generate stableproducer lines. In addition, the invention provides a method for highlyefficient transduction of mammalian cells that have previously beendescribed as difficult to transduce with retroviral constructs. Theinvention also describes the construction of cell lines in which theplasmid-based expression vectors of the invention that direct thesynthesis of retroviral gene products required in trans for virusproduction have been stably integrated into the genome of the producingcells. This invention also describes the construction of retroviralvector plasmids with sequences enabling the episomal persistenceretroviral vectors of the invention without the need for stableintegration of the vector plasmid. All of these stably transfected linescan be used to generate stable cell lines that continuously producerecombinant retrovirus at high titer.

The retroviral constructs are packaging plasmids consisting of at leastone retroviral helper DNA sequence derived from areplication-incompetent retroviral genome encoding in trans all virionproteins required to package a replication incompetent retroviralvector, and for producing virion proteins capable of packaging thereplication-incompetent retroviral vector at high titer, without theproduction of replication-competent helper virus. The retroviral DNAsequence lacks the region encoding the native enhancer and/or promoterof the viral 5′ LTR of the virus, and lacks both the psi functionsequence responsible for packaging helper genome and the 3′ LTR, butencodes a foreign polyadenylation site, for example the SV40polyadenylation site, and a foreign enhancer and/or promoter whichdirects efficient transcription in a cell type where virus production isdesired. The retrovirus is a leukemia virus such as a Moloney MurineLeukemia Virus (MMLV), the Human Immunodeficiency Virus (HIV), or theGibbon Ape Leukemia virus (GALV). The foreign enhancer and promoter maybe the human cytomegalovirus (HCMV) immediate early (IE) enhancer andpromoter, the enhancer and promoter (U3 region) of the Moloney MurineSarcoma Virus (MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3region of Spleen Focus Forming Virus (SFFV), or the HCMV IE enhancerjoined to the native Moloney Murine Leukemia Virus (MMLV) promoter. Theretroviral packaging plasmid may consist of two retroviral helper DNAsequences encoded by plasmid based expression vectors, for example wherea first helper sequence contains a cDNA encoding the gag and polproteins of ecotropic MMLV or GALV and a second helper sequence containsa cDNA encoding the env protein. The Env gene, which determines the hostrange, may be derived from the genes encoding xenotropic, amphotropic,ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virusenv proteins, or the Gibbon Ape Leukemia Virus (GALV env protein, theHuman Immunodeficiency Virus env (gp160) protein, the VesicularStomatitus Virus (VSV) G protein, the Human T cell leukemia (HTLV) typeI and II env gene products, chimeric envelope gene derived fromcombinations of one or more of the aforementioned env genes or chimericenvelope genes encoding the cytoplasmic and transmembrane of theaforementioned env gene products and a monoclonal antibody directedagainst a specific surface molecule on a desired target cell.

Specific embodiments of the retroviral packaging plasmids of theinvention include: pIK6.1MMSVampac, pIK6.1MCVampac, pIK6.1gagpolATG andpIK6.1amenvATG.

The invention includes retroviral vectors that contain a modified 5′LTR, which enables efficient transcription of packagable vectortranscripts in the desired cell type. In addition, the inventionincludes retroviral constructs encoding foreign genes.

In one method of the invention, the packaging plasmids and retroviralvectors are transiently cotransfected into a first population ofmammalian cells that are capable of producing virus, such as humanembryonic kidney cells, for example 293 cells (ATCC No. CRL1573, ATCC,Rockville, Md.) to produce high titer recombinant retrovirus-containingsupernatants. In another method of the invention this transientlytransfected first population of cells is then cocultivated withmammalian target cells, for example human lymphocytes, to transduce thetarget cells with the foreign gene at high efficiencies. In yet anothermethod of the invention the supernatants from the above describedtransiently transfected first population of cells are incubated withmammalian target cells, for example human lymphocytes or hematopoieticstem cells, to transduce the target cells with the foreign gene at highefficiencies.

In yet another method of the invention, the packaging plasmids (eithersingle or double genome) are transiently cotransfected with a retroviralvector plasmid into a first population of mammalian cells, for example293 cells, to produce high titer recombinant retrovirus containingsupernatants.

In still yet another method of the invention, the packaging plasmids arestably expressed in a first population of mammalian cells that arecapable of producing virus, such as human embryonic kidney cells, forexample 293 cells. Retroviral vectors are introduced into cells byeither cotransfection with a selectable marker or infection withpseudotyped virus. In both cases, the vectors integrate. Alternatively,vectors can be introduced in an episomally maintained plasmid. Hightiter recombinant retrovirus-containing supernatants are produced.

The invention further includes mammalian target cells expressing aforeign gene produced by any of the above methods of the invention. Theforeign gene may be a chimeric T cell receptor such as a CD4/zeta orsingle-antibody chain/zeta T cell receptor, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, B and C is a diagrammatic representation of the retroviralpackaging plasmids of the invention used to produce the proteinsnecessary to package retroviral vector transcripts: pIK6.1MMSVampac,pIK6.1MCVampac, pIK6.1gagpolATG, and pIK6.1envATG.

FIG. 2 (Parts A-D) shows the FACS profile of 293 cells transfected withretroviral constructs, as described in Example I, infra.

FIG. 3 (Parts A-B) shows the transduction efficiency determined bySouthern blot analysis of infected 3T3 DNA, as described in Example I,infra.

FIG. 4 is a bar graph of the data from experiments in which CD8+ T cellswere transduced by, first, transient transfection of 293 cells witheither pRTD2.2F3 or pRTD2.2F15 and pIK6.1MCVampac, followed bycocultivation of the 293 cells with the CD8+ T cells and analysis oftransduction efficiency by FACS, as described in Example II, infra.

FIG. 5 (Parts A-D) shows the results of FACs analysis of hematopoieticstem cells transduced with the KAT packaging constructs andcocultivation with 293 cells, as described in Example III, infra.

FIG. 6 examines whether the cocultivation of CD34+ cells with KATtransfected 293 cells leads to high efficiency transduction as analyzedby Southern blotting, as described in Example III, infra.

FIG. 7 compares the transduction efficiency of CD34+ cells transduced bythe KAT system to that of cocultivation with a stable PA317 producer bySouthern blotting, as described in Example III, infra.

FIG. 8 (Parts A-E) shows the results of FACs analysis of human CD34+hematopoietic progenitors transduced with the KAT pIKT retrovirus vectorconstructs following transfection of 293 cells with pIKT vectors andcocultivation, as described in Example IV, infra.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

The present invention provides novel optimized transient expressionplasmids (designated “KAT”) for production of retroviral virions inwhich high steady state levels of retroviral packaging functions andpackagable vector transcripts are produced following introduction of KATplasmids into mammalian cells capable of efficient transienttransfection and expression, in the absence of plasmid replication ofviral vector and packaging function plasmids. The absence of plasmidreplication enables production of high titer virions while minimizingthe potential for production of replication competent retrovirus byrecombination. Use of the KAT system yields 10-30 fold higher viraltiters compared to cotransfection of packaging functions and vectorplasmids into COS cells, as described by Landau and Litman, J. Virol. 66(8): 5110-5113 (1992)). Alternatively, because the KAT packagingfunction and viral vector plasmids contain the SV40 origin ofreplication, they can be transfected into cell lines that enablereplication of SV40 origin-containing plasmids due to expression of theSV40 T antigen, such as tsa201 (Heinzel et al., J. Virol. 62 (10):3738-3746 (1988)). Using the KAT system, viral titers in the presence ofplasmid replication are 3 to 10-fold higher than in the absence ofreplication. Whether replicating or nonreplicating plasmids are used,the KAT system permits the rapid production of high titer recombinantretrovirus supernatants without the need for generating stable producerlines.

The retroviral constructs of the invention also find use in the methodof the invention to transduce by cocultivation or by supernatantinfection, with high efficiency, mammalian cells, such as primary humancells, that are typically refractory to transduction by conventionalmeans.

The plasmids of the invention also find use in the construction ofstable cell lines that constitutively produce the retroviral proteinsrequired in trans for the production of retrovirus particles: gag, poland env. These stable packaging constructs can be introduced into humancell lines by calcium phosphate transfection or electroporation,together with a dominant selectable marker, such as neo, DHFR*, GlnSynthetase, ADA, followed by selection in the appropriate drug andisolation of clones. This enables the production of high titer stableproducer clones following introduction of a retroviral construct intothese cells. These cell lines have all of the same properties of thetransiently transfected producer cells. However, due to stableintegration of both packaging function and virus vector, they continueto produce high titer retrovirus indefinitely in the absence of drugselection.

Plasmids containing the packaging functions can be split with oneencoding the gag and pol genes and a second encoding the env geneproduct. Packaging lines containing two viral genomes have beendescribed (Bosselman et al., Molec. Cell. Biol. 7 (5): 1797-1806 (1987);Markowitz et al., J. Virol. 62 (4): 1120-1124 (1988); Danos andMulligan, Proc. Natl. Acad. Sci. (USA) 85:6460-6464 (1988)) and aredesirable due to the significantly reduced chance for the generation ofreplication competent retrovirus (RCR) following recombination between aretroviral vector and packaging construct. Use of the plasmids of theinvention results in a packaging line yielding the high efficiencytransduction of the transient system. The novel plasmids of theinvention enable a significant advance over previously described twogenome packaging lines. The KAT plasmids encoding gagpol and env geneshave been constructed so that only protein coding sequence from theretroviral genome is present. Using the retroviral vectors described inthe invention, no overlap exists between the retrovirus vector andpackaging genomes at their 3′ ends. This structure in combination withreplacement of the gag start codon (ATG) in the vector with a stop codonabsolutely precludes the generation of replication competent retrovirusin contrast to previously described packaging lines where complete viralgenomes containing mutations (Danos and Mulligan, Proc. Nat'l. Acad.Sci. (USA) 85:6460-6464 (1988)) or deletions (Bosselman et al., Molec.Cell. Biol. 7 (5): 1797-1806 (1987); Markowitz et al., J. Vir ol. 62(4): 1120-1124 (1988)). These prior known packaging lines containoverlap at the 3′ end of the virus vector with the packaging line andcan potentially generate RCR.

Two genome packaging lines are constructed by sequential introduction ofthe gagpol plasmid followed by the env-containing plasmid. The env genesare responsible for recognition of cell surface receptors. Fivefunctionally and structurally different env genes have been identifiedin murine leukemia viruses and have been shown to have geneticallydistinct receptors (Battini et al., J. of Virol. 66:1468-1475 (1992)).Human host range with murine leukemia virus vectors is possible by theintroduction of the amphotropic env gene into a cell line that expressesthe ecotropic MLV gagpol Danos and Mulligan, Proc. Nat'l. Acad. Sci. USA85:6460-6464 (1988)). The xenotropic and 10A1 MLV viruses have humanhost range, as well as the gibbon ape leukemia and feline leukemiaviruses. Using cDNA clones of these env genes, one or more can be stablyintroduced into a gagpol line to create a packaging line where theretrovirus produced following introduction of a retroviral vector canenter the target through multiple genetically distinct receptors. Thisleads to substantial increases in apparent viral titer. The vectors ofthe invention provide for the ability to create these types of novelpackaging lines.

The expression plasmids for gag/pol and env contain a functional poly Aaddition signal (poly A site) which is essential for transcriptiontermination by RNA polymerase II (Connelly and Manley Genes Dev.2:440-452 (1988)). The poly A site may be derived from a viraltranscription unit, a cellular gene, or a synthetic oligonucleotide.Examples of viral poly adenylation sites include the SV40 early regionpoly A site (Fitzgerald and Shenk, Cell 24:251-260 (1981)) or thehepatitis B surface antigen poly A site (Simonsen and Levinson, Mol.Cell., Bio. 3:2250-2258 (1983)) Examples of polyadenylation signalsderived from cellular genes include human pro alpha 2 (1) collagen(Myers et. al., J. Biol. Chem. 258:10128-10135 (1983)), bovine growthhoremone (Woychik et al., Proc. Natl. Acad. Sci., USA 81:3944-3988(1984)) and the human alpha globin gene (Orkin et. al., 4:453-456(1985)). An example of an efficient synthetic polyadenylation site isthe sequence AATAAA(N) 22-23 (GT)n(T)N (Levitt et. al., Genes Dev.3:1019-1025 (1989)). One skilled in the art may substitute any of theabove polyadenylation signals for the SV40 poly A signal used in theinstant plasmids by using conventional techniques.

The techniques used to construct vectors, and transfect and infectcells, are widely practiced in the art, and most practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures. However, for convenience, the followingparagraphs may serve as a guideline.

Construction of the vectors of the invention employs standard ligationand restriction techniques which are well understood in the art (seeManiatis et al., in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y. (1982)). Isolated plasmids, DNA sequences, orsynthesized oligonucleotides are cleaved, tailored, and religated in theform desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. (See,e.g. New England Biolabs, Product Catalog.) In general, about 1 μg ofplasmid or DNA sequences is cleaved by one unit of enzyme in about 20 μlof buffer solution. Typically, an excess of restriction enzyme is usedto insure complete digestion of the DNA substrate. Incubation times ofabout one hour to two hours at about 37° C. are workable, althoughvariations can be tolerated. After each incubation, protein is removedby extraction with phenol/chloroform, and may be followed by etherextraction, and the nucleic acid recovered from aqueous fractions byprecipitation with ethanol. If desired, size separation of the cleavedfragments may be performed by polyacrylamide gel or agarose gelelectrophoresis using standard techniques. A general description of sizeseparations is found in Methods of Enzymology 65:499-560 (1980).

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 minutes at 20° C. in 50 mM Tris (pH 7.6) 50 mM NaCl, 6 mMMgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at 5′sticky ends but chews back protruding 3′ single strands, even though thefour dNTPs are present. If desired, selective repair can be performed bysupplying only one of the dNTPs, or with selected dNTPs, within thelimitations dictated by the nature of the sticky ends. After treatmentwith Klenow, the mixture is extracted with phenol/chloroform and ethanolprecipitated. Treatment under appropriate conditions with Sl nuclease orBal-31 results in hydrolysis of any single-stranded portion.

Ligations are performed in 15-50 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 mg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) unites T4 DNA ligase at 0° C. (for “sticky end” ligation) or 1mM ATP, 0.3-0.6 (Weiss) unites T4 DNA ligase at 14° C. (for “blunt end”ligation). Intermolecular “sticky end” ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 mM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 μM total endsconcentration.

The retroviral vectors and packaging plasmids of the KAT system areprepared as follows:

Production of Novel Retroviral Vectors and Packaging Plasmids

The KAT constructs include DNA packaging plasmids consisting of at leastone retroviral helper DNA sequence derived from areplication-incompetent retroviral genome, e.g. a leukemia virus genome,encoding in trans all virion proteins required to package a replicationincompetent retroviral vector, and for producing virion proteins capableof packaging the replication-incompetent retroviral vector at hightiter, without the production of replication-competent helper virus. Inone embodiment the retroviral packaging DNA sequence lacks the regionencoding the native enhancer and/or promoter of the viral 5′LTR of thevirus, and lacks the psi function sequence responsible for packaginghelper genome as well as the 3′ LTR, but encodes a foreign enhancerand/or promoter which directs efficient transcription in a cell typewhere virus production is desired, and includes an SV40 polyadenylationsite. The transcription initiation site of the foreign enhancer andpromoter is joined to the leukemia virus genome at the 5′ end of the “R”region of the 5′ LTR.

The retrovirus may be a Moloney Murine Leukemia Virus (MMLV), the HumanImmunodeficiency Virus (HIV) or the Gibbon Ape Leukemia virus (GALV).The foreign enhancer and promoter joined to the R region of the 5′ LTRmay be the human cytomegalovirus (HCMV) immediate early (IE) enhancerand promoter (the U3 region) of the Moloney Murine Sarcoma Virus (MMSV),the U3 region of the Rous Sarcoma Virus (RSV), the U3 region of theSpleen Focus Forming Virus (SFFV), or the HCMV IE enhancer joined to thenative Moloney Murine Leukemia Virus (MMLV) promoter.

All psi (ψ)-packaging plasmids are derivatives of the plasmid pIK1.1.pIK1.1 is a mammalian expression vector constructed by four successiveinsertions into pMF2, which is created by inserting the syntheticpolylinker 5′-HindIII-SphI-EcoRI-AatII-BglI-XhoI-3′ into KpnI and SacIsites of pSKII (Stratagene, San Diego, Calif.), with loss of the Kpn Iand Sac I sites. First, a BamHI-XbaI fragment containing the SV40 Tantigen polyadenylation site (nucleotides 2770 to 2533 of SV40, Reddy etal., Science 200:494-502 (1978)) and an NheI-Sal I fragment containingthe SV40 origin of replication (nucleotides 5725 to 5578 of SV40) areinserted by three-part ligation between the Bg1II and XhoI sites, withthe loss of the Bg1II, BamHI, XbaI, NheI, Sa1I and XhoI sites. TheseBamHI-XbaI and NheI-Sa1I fragments are synthesized by PCR with pSV2neo(Southern and Berg, J. Mol. Appl. Gen. 1:327-341 (1982)) as the templateusing oligonucleotide primer pairs 3 and 4, and 5 and 6, respectively,which incorporated BamHI, XbaI, NheI and Sa1I sites at their respectiveends. Second, an SphI-EcoRI fragment containing the splice acceptor ofthe human α1 globin gene second exon (nucleotides +143 to +251) isinserted between the SphI and EcoRI sites. This SphI-EcoRI fragment issynthesized by PCR with pπSVαHP (Treisman et al., Proc. Natl. Acad. Sci.USA 80:7428-7432 (1983)) as the template using oligonucleotide primers 7and 8, which incorporate SphI and EcoRI sites at their respective ends.Third, the synthetic polylinker 5′-EcoRI-Bg1II-NcoI-ApaI-AatII-3′ isinserted between the EcoRI and the AatII sites. Fourth, a HindIII-SacIfragment containing the CMV IE enhancer/promoter (nucleotides −674 to−19, Boshart et al., Cell 41:521-530 (1985)) and a chemicallysynthesized SacI-SphI fragment containing the CMV IE first exon/splicedonor (nucleotides −19 to +170) are inserted by three-part ligationbetween the HindIII and SphI sites. The HindIII-SacI fragment isprepared by PCR with pCDM8 (Seed, Nature 329:840-842 (1987); Seed andAruffo, Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987)) as the templateusing oligonucleotide primers 9 and 10, which incorporated HindIII andSacI sites at their respective ends.

(SEQ ID NO. 1) Primer 3: 5′-GGTCGACCTGGATCCGCCATACCACATTTGTAG-3′ (SEQ IDNO. 2) Primer 4: 5′-GCCGCGGCTCTAGAGCCAGACATGATAAGATAC-3′ (SEQ ID NO. 3)Primer 5: 5′-AAGCTTGTGCTAGCTATCCCGCCCCTAACTCCG-3′ (SEQ ID NO. 4) Primer6: 5′-CGAAATCGGTCGACCGCAAAAGCCTAGGCCTCC-3′ (SEQ ID NO. 5) Primer 7:5′-GTCTATAGCATGCTCCCCTGCTCCGACCCG-3′ (SEQ ID NO. 6) Primer 8:5′-GGTACCGAATTCTCCTGCGGGGAGAAGCAG-3′ (SEQ ID NO. 7) Primer 9:5′-CGCCAAGCTTGGCCATTGCATACGGT-3′ (SEQ ID NO. 8) Primer 10:5′-GAGGTCTAGACGGTTCACTAAACGAGCTCT-3′

An Xba I site is introduced at the transcription initiation site of theHCMV IE promoter in pIK1.1 by replacement of the chemically synthesizedSac I/Sph I oligonucleotide encoding −19 to +170, described above, witha chemically synthesized Sac I/Sph I oligonucleotide where an Xba I siteat nucleotides +1 to +6 had been introduced to generate pIK6.1. Thisallows insertion of any enhancer/promoter as a Hind III to Xba Icassette so as to insert the appropriate enhancer and promoter that willdirect the highest possible expression level of the desired sequences inthe desired cell type. In order to obtain the highest expression levelsin mouse fibroblast NIH 3T3 (ATCC CRL 1658) or M. dunni (ATCC CRL2017),the complete MMSV U3 region was synthesized by PCR using the plasmid pN7(Miller et al., Mol. Cell. Biol. 5:431-437 (1985)) as a template and twoprimers: one which encoded a HindIII site and the 5′ 21 nucleotides ofthe U3, and a second which encoded the 3′ 21 nucleotides of the MMLV U3region and an Xba I site. This PCR fragment was cloned between theHindIII and Xba I sites of pIK6.1 to generate pIK6.1MMSV. In order todirect high level expression in human cells, pIK6.1MCV was constructedby isolation of the Nco I/Spe I fragment of the HCMV IE enhancer(Boshart et al., supra), addition of synthetic oligonucleotide Bcl Ilinkers, and insertion in the Bam HI site of the plasmid pΔHB (Dr. P.Robbins, University of Pittsburgh, Pittsburgh, Pa.). This plasmid wasdesignated pMCV. pΔHB is a plasmid in which the ClaI to EcoRI fragmentof pZIPneoSVX (Cepko et. al, supra), containing viral sequencesincluding the 3′ LTR, has been cloned into the ClaI and Eco RI sites ofpBR322 and where the Sau 3AI to Hpa II enhancer fragment of MMLV U3 hasbeen removed. Due to the homology between the MMLV U3 and the MMSV U3,the PCR primers described above were used to generate a Hind III/Xba Ilinker fragment encoding the U3 fragment of pMCV, which was cloned intopIK6.1 to generate pIK6.1MCV. These plasmids, as well as pIK6.1, werefurther modified by deletion of 112 nucleotides of the SV40polyadenylation site between the ApaI site at the 3′ end of the pIKpolylinker and the Hpa I site in the SV40 polyadenylation site andreplacement with an Nhe I linker to create pIK6.1.Nhe, pIK6.1.MMSV.Nheand pIK.6.1MCV.Nhe.

pIK6.1MMSVampac and pIK6.1MCVampac were constructed by insertion of 3813base Sac I/Sal fragment encoding a portion of the U3 region, the R, andU 5 regions, the gag gene and a portion of the pol gene of pMOV psi-(Mann et al., supra), and the 4140 base pair Sal 1-Nhe I fragmentencoding pol/env, derived from pCRIPamgag-2 (Danos and Mulligan, Proc.Natl. Acad, Sci. USA, 85:6460-6464 (1988) between the Sac I and Nhe Isites of pIK6.1MMSV.Nhe or pKI6.1MCV.Nhe, respectively. pCRIPamgag-2 isa derivative of pCRIPamgag where the pBR322 plasmid backbone has beenreplaced by the plasmid pUC19. The resulting plasmids encode the gag andpol genes from ecotropic MMLV and the envelope gene from the 4070Aamphotropic MLV (Chattopadhyay et al., J. Virol. 39 (3): 777-791 (1981))and are diagramed in FIG. 1A.

To delete untranslated sequences 3′ from the envelope gene ofpIK6.1MCVampac a PCR reaction was performed using pIK6.1MCVampac as thetemplate with synthetic oligonucleotides 5′ CTGATCTTACTCTTTGGACC3′(SEQID NO. 31) and 5′GAATTCGCTAGCCTATGGCTCGTACTCTATAG 3′(SEQ ID NO. 32). Theresulting 142 basepair PCR product was cut with ClaI and NheI. This 100base pair fragment was excised and used to replace the corresponding 172base pair ClaI to NheI fragment of pIK6.1MCVampac to give pIK6.1MCVampacUTΔ.

pIK6.1amenvATGUTΔ was constructed by replacing the 172 base pair ClaI toNheI fragment in pIK6.1amenvATG with the 100 base pair ClaI to NheIfragment from pIK6.1MCVampacUTΔ. pIK6.1MCVamenvATGUTΔ was constructed byreplacing the 961 base pair Hind III to Eco RI fragment containing theCMV promotor and alpha globin splice acceptor in pIK6.1amenvATGUTΔ withthe corresponding 896 base pair Hind III to Eco R1 fragment frompIK6.1MCV.

pIK6.1MCVgagpolATG was constructed by replacing the 961 base pair HindIII to Eco RI fragment containing the CMV promotor and alpha globinsplice acceptor in pIK6.1gagpolATG with the corresponding 896 base pairHind III to Eco R1 fragment from pIK6.1MCV.

The retroviral packaging plasmids of the invention, designatedpIK6.1MMSVampac and pIK6.1MCVampac, have been deposited with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., under the Budapest Treaty, and have there beenidentified as follows:

Plasmid ATCC Accession No. Deposit Date pIK6.1MMSVampac 75484 Jun. 11,1993 pIK6.1MCVampac 75483 Jun. 11, 1993

In another embodiment, the packaging functions may be encoded by twoplasmid based expression vectors, for example two helper sequences,where a first helper sequence contains a cDNA encoding the gag and polproteins of ecotropic MMLV and a second helper sequence contains a cDNAencoding a retroviral env protein. The Env gene, which determines thehost range, may be derived from the genes encoding the xenotropic,amphotropic, ecotropic, polytropic (mink focus-forming) or 10A1 murineleukemia virus, Gibbon Ape Leukemia Virus (GALV), the HumanImmunodeficiency Virus (gp160) env proteins; the Vesicular StomatitusVirus (VSV) G protein; the Human T cell leukemia (HTLV) type I and IIenv gene products; a chimeric envelope gene derived from combinations ofone or more of the aforementioned env genes; or chimeric envelope genesencoding the cytoplasmic and transmembrane of the aforementioned envgene products and a monoclonal antibody directed against a specificsurface molecule on a desired target cell.

Construction of plasmids which reflect this embodiment is described asfollows: pIK6.1gagpolATG, encoding the MMLV gag and pol genes, wasconstructed first by digestion of pMOVpsi- with Sca I, addition of a NheI synthetic linker, redigestion with Afl II and isolation of the 5.2 kbAfl II/Nhe I fragment (nucleotides 644 to 5869 of MMLV). A syntheticoligonucleotide encoding nucleotides 621 to 644 of MMLV (ATG of the gaggene to Afl II), in which the ATG at nucleotide 621 was converted to aNco I site, was ligated together with the Afl II/Nhe I fragment betweenthe Nco I site polylinker and the Nhe I site at the 5′ end of the SV40poly adenylation site of pIK6.1Nhe.

pIK6.1amenvATG, encoding the MLV 4070A Env gene, was constructed bydigestion of pCRIPAMGAG-2 (Danos and Mulligan, supra) with Afl 111 andredigestion with either Nhe1 or HinP1 and isolation of the 0.325 kb HinP1/Afl 111 fragment (nucleotides 37 to 365 of the MLV 4070A Env gene;(Ott et. al., J. Virol. 64 (2): 757-766 (1990)) and the 1.7 kb Afl111/Nhe 1 fragment (from nucleotide 365 of the MLV 4070A Env gene; (Ottet. al., supra) to the Nhe 1 site in the MMLV 3′ LTR of pCRIPAMGAG-2(Danos and Mulligan, supra) respectively. A synthetic oligonucleotideencoding nucleotides 37 to 43 of the MLV 4070A Env gene (ATG of the envgene to HinP 1), in which the ATG at nucleotide 37 was converted to aNco I site, was ligated together with the HinP 1/Afl 111 fragment andthe Afl 111/Nhe 1 fragment between the Nco I site in the polylinker andthe Nhe I site at the 5′ end of the Sv40 poly adenylation site ofpIK6.1Nhe. These plasmids are diagramed in FIG. 1A.

The two genome retroviral packaging plasmids of the invention,designated pIK6.1gagpolATG and pIk6.1amenvATG, have been deposited withthe American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., under the Budapest Treaty, and have there beenidentified as follows:

Plasmid ATCC Accession No. Deposit Date pIK6.1gagpo1ATG 75486 Jun. 11,1993 pIK6.1amenvATG 75485 Jun. 11, 1993Retroviral Vectors

Both single genome and two genome packaging constructs utilizeretroviral vectors that contain modified 5′ LTRs that direct efficienttranscription in the cell type where retrovirus is to be produced. Theretroviral vectors of the invention are modeled after pZen (Johnson etal., The EMBO Journal 8 (2): 441-448 (1989)), a neo-version ofpZIPneoSVX (Cepko et al., Cell 37:1053-1062 (1985)), in which the geneproduct to be expressed is cloned downstream of the splice acceptor inthe position normally occupied by the neo cassette (Cepko et al.,supra). In addition, viral gag sequences up to the Nar I site of MMLV(nucleotide 1038) were added for improved packaging (Armentano et al.,J. Virol. 61:11647-1650 (1987)) and the Xho I to Cla I fragment ofpZIPneoSVX was deleted (Cepko et al., supra). The Eco RI to Apa Ipolylinker from pIK1.1 was inserted downstream of the splice acceptor toenable transfer of inserts from pIK plasmids into retroviral constructs.The resulting plasmid is designated pRTD1.2 and contains both 5′ and 3′MMLV LTRs. The 5′ LTR U3 region of pZIPneoSVX was replaced with the MMSVU3, derived from the HindIII/Sac I fragment of pIKMMSV, to generatepRTD4.2. In pRTD2.2, the U3 region of the 5′ LTR of pZIPneoSVX wasreplaced with the Hind III/Sac I fragment from pIK1.1 encoding the CMVimmediate early enhancer/promoter, which was fused to the MMLV R regionby an oligonucleotide that encodes nucleotides 19 (Sac I) to +1 of theHCMV promoter linked to nucleotides +1 to +32(KpnI) of MMLV (Schinnicket al., Nature 293:543-548 (1980)). pRTD2.2SVG was constructed byreplacement of the (750 base pair) Sac I to Bst EII fragment of pRTD2.2with the (736 base pair) Sac I to Bst EII fragment of LXSN (Miller andRosman, BioTechniques 7:980-990 (1989)). pRTD2.2SSA was constructed byreplacement of the (1441 base pair) Sac I to Eco RI fragment of pRTD2.2with the (1053 base pair) Sac I to Eco RI fragment of LXSN (Miller andRosman, supra). pRTD2.2SVGE- was constructed by synthesis of anoligonucleotide encoding nucleotides 2878-2955 of pLXSN (GenBankAccession Bank, M28248) which had been appended by addition of an Apa Isite on it's 5′ end. This was used to replace the Apa I to Nhe Ifragment of pRTD2.2SVG, which contains the DNA sequence 3′ of the of thepolylinker and 5′ of the Nhe I site in the 3′ LTR. These retroviralvector constructs of the invention have a pBR322 backbone and includepRTD2.2, pRTD4.2, pRTD2.2SVG, pRTD2.2SVGE- and pRTD2.2SSA.

In order to permit plasmid replication in cells which express the SV40 Tantigen, the sequences between the 5′ and 3′ LTRs of pRTD2.2 were clonedbetween the SacI and Eco RI sites of pIK1.1, described above, whichcontains the SV40 origin of replication to form vector pIKT2.2.pIKT2.2SVG was constructed by insertion of the fragment defined at its5′ end by the Sac I site in the HCMV promoter of pRTD2.2SVG and definedat its 3′ end by an Eco RI site located 750 base pair downstream of the3′ LTR of pRTD2.2SVG, between the SacI and Eco RI sites of pIK1.1.pIKT2.2SVGE-F3 was constructed by replacing the 182 base pair ApaI toNheI fragment of pIKT2.2SVGF3 with the 80 base pair ApaI to NheIfragment from pRTD2.2SVGE-F3 as described above.

pRT43.2F3 was derived from pIKT2.2SVGE-F3 by replacing the Eco RI toApaI polylinker located approximately 750 base pairs downstream from the3′ LTR with a synthetic oligonucleotide containing an AscI recognitionsite. In addition, the Nde I site at the 3′ end of the viral gagsequences has been converted to an XhoI site by oligonucleotideinsertion. pRT43.3PGKF3 was derived from pRT43.2F3 first by removal ofthe 3′ LTR in pRT43.2F3 and insertion of a 3′ LTR in which the sequencesfrom PvuII to XbaI were deleted (MMLV, GenBank session #J02255nucleotide numbers 7938-8115). In addition the MMLV splice acceptorregion has been replaced with the human phosphoglycerate kinase genepromotor (GenBank Accession #M11958 nucleotides 2-516) which was clonedinto a polylinker with a XhoI site at its 5′ end and an Eco RI at its 3′end.

In one embodiment of the retroviral vectors of the invention, DNAencoding genes to be transduced into mammalian target cells using themethod of the invention, for expression of chimeric receptor constructsare prepared. The construction of the chimeric receptor constructs isdescribed below.

CD4/CD3 Zeta and Anti-HIV/CD3 Zeta Retroviral Vectors

KAT retroviral vectors pRTD2.2F3, pRTD2.2SVGF3, pRTD2.2SSAF3,pRTD2.2SVGF3E-, pIKT2.2SVGF3 were constructed by Eco RI/Apa I digestionof pIKF3 (described below), isolation of the 1.9 kb fragment, followedby ligation of this fragment between the Eco RI and Apa I sites in thepIK polylinker of the vectors pRTD2.2, pRTD2.2SVG, pRTD2.2SSAF3,pRTD2.2SVGE-, pIKT2.2SVG. KAT retroviral vector pRTD2.2F15 wasconstructed by Eco RI/Apa I digestion of pIKF15neo (described below),isolation of the 2.2 kb fragment, followed by ligation of this fragmentbetween the Eco RI and Apa I sites in the pIK polylinker of the vectorpRTD2.2. These vectors encode a chimeric molecule containing theextracellular domain of human CD4 (F3 derivatives) or a single chainantibody against gp41 of HIV (F15 derivatives), respectively, fused tothe cytoplasmic domain of the CD4 receptor (amino acids 372-395 of themature CD4 chain) and the transmembrane domain of the CD3-complexassociated-gene zeta (Δ) (amino acids 372-395 of the mature zeta chain).Chimeric receptor cassettes encoding either the extracellular domains(residues 1-371 of the mature CD4 protein) of the human CD4 receptor(designated F3) or a single chain antibody to HIV gp41 derived from ahuman antibody (98.6) specific for the gp41 moiety of the HIV envelopeprotein (designated F15) were fused to the CD3 Δ chain and clonedbetween the Eco RI and Apa I sites of pIK1.1 described above. In thesingle-chain antibody, the variable domains of both the heavy and lightchain genes were covalently linked via a peptide tether, to create anantigen binding site on a single molecule.

A more detailed description of the construction of the chimericreceptors follows.

Construction of CD4-zeta Chimeras

Plasmid pGEM3zeta bears the human zeta cDNA (Weissman et al., Proc.Natl. Acad. Sci. USA 85:9709-9713 (1988). The plasmid pBS.L3T4 bears thehuman CD4 cDNA (Littman and Gettner, Nature 325:453-455 (1987)). ABamHI-ApaI restriction fragment (approximately 0.64 kb) encompassing theentire human zeta chain coding sequence from residue 7 of theextracellular (EXT) domain, was excised from pGEM3zeta, and subclonedinto the BamHI and ApaI restriction sites of the polylinker ofpBluescript II SK (+) 9pSK is a phagemid based cloning vector fromStratagene (San Diego, Calif.), generating pSK.zeta. Subsequently, aBamHI restriction fragment encompassing the entire CD4 coding sequence(approximately 1.8 kb) was excised from pBS.L3T4, and subcloned into theBamHI site of pSK.zeta, generating pSK.CD4.zeta.

Single-stranded DNA was prepared from pSK.CD4.zeta (StratagenepBluescript II protocol), and used as a template foroligonucleotide-mediated directional mutagenesis (Zoller and Smith,Nucleic Acids Res. 10:6487-6500 (1982)) in order to generate CD4-zetachimeras with the desired junctions described below. CD4-zeta fusions 1,2, and 3 were subsequently sequenced via the Sanger dideoxynucleotidetechnique (Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467 (1977)),excised as EcoRI-ApaI restriction fragments, and cloned into thepolylinker of expression vector pIK.1.1 or pIK.1.1.N at identical sites.

An EcoRI-BamHI restriction fragment (approximately 1.8 kb) encompassingthe entire coding region of CD4 was excised from pSK.CD4.zeta, andsubcloned between the EcoRI and Bg1II sites of the pIK.1.1 orpIK.1.1.Neo polylinker.

The plasmid pUCRNeoG (Hudziak, et al., Cell (1982) 31:137-146) carriesthe neomycin gene under the transcriptional control of the Rous Sarcomavirus (RSV) 3′ LTR. The RSV-neo cassette was excised from PURCNeoG as aHincII restriction fragment (approximately 2.3 kb), and subclonedbetween the two SspI sites of pIK.1.1, generating pIK.1.1.Neo.

The CD4-zeta chimeric receptor F3 was constructed from the extracellular(EC) and cytoplasmic (CYT) domains of CD4 and zeta respectively. Thetransmembrane (TM) domain of this receptor was derived from CD4. F3retains the CD4 EXT domain comprising all four V domains (residues 1-371of the mature CD4 protein), the TM domain of CD4 (residues 372-395 ofthe mature CD4 chain), and the CYT domain of zeta (residues 31-142 ofthe mature zeta chain).

Preparation of Single Chain Antibody-Zeta Chimeric Receptor

Construction of expression vector encoding the heavy chain of humanmonoclonal antibody (mAb) 98.6:

To direct the expression of the heavy chain of human mAb 98.6 (S.Zolla-Pazner, Proc. Natl. Acad. Sci. (1989) 86:1624-1628), the plasmidpIK.98.6-γFL was constructed. A full length IgG1 heavy chain cDNA wasgenerated by reverse transcription of 5 μg of total RNA from the cellline SP-1/98.6 (Zolla-Pazner, supra) using oligo-dT as the primer,followed by PCR using oligonucleotide primers 17 and 2 (see below). The1.5 kb Eco RI to Bg1II fragment was cloned between the Eco RI and Bg1IIsites of pIK1.1. To ensure that the heavy chain would be of the desiredallotype, the Kas I-Bg1II fragment of the cDNA was replaced with a 0.94kb Kas I-Bg1II fragment from pIK.Cγ1. pIK.Cγ1 was constructed by theinsertion of a cDNA coding for the constant region of IgG1 heavy chainobtained by PCR using DNA from a human spleen cDNA library (Clontech,Inc., Palo Alto, Calif.) as substrate and oligonucleotide primers 2 and18 (see below), between the Eco RI and Bg1II sites of pIK1.1.

Construction of expression vector encoding the light chain of humanmonoclonal antibody (mAb) 98.6:

To direct the expression of the light chain of mAb 98.6, the plasmidpIK.98.6κFL was constructed. A full length IgG1 light chain cDNA wasgenerated by reverse transcription of 5 μg of total RNA from the cellline SP-1/98.6 using pdN₆ (Pharmacia/LKB) as the primer, followed by PCRwith primers 19 and 20 (see below). The 0.78 fragment was then cut withEco RI and Bg1II and cloned between the Eco RI and Bgl II sites ofpIK1.1.

Construction of expression vector encoding SAb derived from the heavyand light chains of mAb 98.6:

a) Construction of pIK98.6-K/L/H:

To direct the expression of a single-chain antibody (SAb) form of mAb98.6, pIK.98.6-K/L/H was constructed. The SAb expressed consists of thesecretion leader sequence and amino acids 1-107 of the mature 98.6 mAblight chain variable (V_(L)) region fused to a 14 amino acid linker ofthe sequence GSTSGSGSSEGKG (SEQ ID NO. 9) (L212, Betzyk et al., J. Biol.Chem. (1990) 265:18615-18620), which in turn was fused to amino acid 1of the mature 98.6 mAb heavy chain V_(H) region. This was then fused atamino acid 113 to amino acid 234 of the IgG1 heavy chain constantregion, in order to delete the CH1 domain of the IgG1 heavy chainconstant region for improved secretion. pIK.98.6-K/L/H was constructedin three steps.

First, deletion mutagenesis was performed to fuse amino acid 113 of theV_(H) region of mAb 98.6 to amino acid 234 of the IgG1 heavy chain,using the single stranded template form of pIK.98.6-γFL as the templateand oligonucleotide 21 as primer (see below). Correctly deleted plasmidswere found using oligonucleotide 22 as a probe (see below). This plasmidis referred to as pIK.H/Fc-int. To fuse amino acid 107 to the aminoterminus of the linker peptide, the V_(L) region of the mAb 98.6 lightchain was generated by PCR using pIK.98.6-κFL as substrate andoligonucleotides 23 and 24 as primers (see below). This was done toplace a Sal I site at the 3′ end of the V_(L) sequence, without alteringthe amino acid sequence of the resulting protein. This fragment,together with oligonucleotides 25 and 26 (see below) was ligated betweenthe EcoRI and Bg1II sites of pIK1.1, generating the plasmid pIK.K/L-int.

In the final step, the 0.45 kb fragment of pIK.K/L-int was clonedbetween the Eco RI and Kpn I sites of pIK.H/Fc-int., generating plasmidpIK.K/L/H-int. Single-stranded DNA from this plasmid was used astemplate and oligonucleotide 27 was used as primer (see below) to fusethe carboxy-terminal amino acid of the linker to amino acid 1 of theV_(H) region of mAb 98.6 by deletion mutagenesis. Correctly deletedplasmids were found using oligonucleotide 28 as a probe (see below). Theresulting plasmid is pIK.98.6K/L/H.

b) Construction of pIK.CD4γ2:

The plasmid pIK.CD4γ2 was constructed to direct the expression of afusion protein composed of the secretion leader and the first 180 aminoacids of the mature CD4 antigen fused to amino acid 234 of the humanIgG2 heavy chain constant region and thus containing part of the hingeand all of the CH2 and CH3 domains. This deletes the CH1 domain of theIgG2 heavy chain for improved secretion. pIK.CD4γ2 was constructed bygenerating a fragment containing the Fc portion of the human IgG2 heavychain by PCR using DNA from a human spleen cDNA library (Clontech) assubstrate and oligonucleotides 3 and 4 as the primers. The 0.75 kb Nhe Ito Bgl II fragment generated was ligated together with the 0.6 kb Eco RIto Nhe I fragment from pSKCD4Δ between the Eco RI and Bgl II sites ofpIK1.1.

c) Construction of pIK.F5:

The plasmids pIK.F7 was constructed to direct expression of severalversions of CD4/IgG/zeta (Δ) fusion proteins which all contain a humanmembrane-bound IgG membrane hinge domain (Tyler et al. (1982) Proc.Natl. Acad. Sci. 79:2008-2012). Each protein to be expressed containedamino acids 1-180 of CD4 receptor, followed by amino acids 234-445 ofhuman IgG2 heavy chain constant region, followed by the 18 amino acid M1membrane hinge domain of human IgG3 (Bensmana and

Construction of pIK.F15neo:

To direct the expression of a fusion protein consisting of the K/L/H SAbform of mAb 98.6 linked at amino acid 445 of the IgG1 heavy chain to the18 amino acid IgG3 M1 membrane hinge, which was in turn fused to the CD4transmembrane domain (amino acids 372-395) and Δ cytoplasmic domain(amino acids 31-142), pIK.F15neo was constructed by inserting the 1.5 kbNsi I fragment of pIK.98.6-K/L/H between the Nsi I sites of pIK.F7neoand a clone of the correct orientation was selected.

Production of Retrovirus in Mammalian Cells

Single or double genome KAT packaging plasmids, for examplepIK6.1MMSVampac,pIK6.1MCVampac, or pIk6.1amenvATG and pIK6.1gagpolATG(all described above), together with KAT retroviral constructs, forexample, but not limited to pRTD2.2F3, pRTD2.2SVGF3, pRTD2.2SSAF3,pRTD2.2SVGF3E-, pIKT2.2SVGF3, pRTD2.2F15 (as described above), preparedas described above, are introduced into mammalian cells that can producevirus by standard means such as calcium phosphate cotransfection (Wigleret al., Proc. Natl. Acad. Sci. USA 76:1373-1377 (1979)). Mammalian cellsthat can produce virus and that may be transfected by the KAT constructsof the invention include, but are not limited to, human embryonic kidneycells such as 293 cells, tsa201 cells, mouse 3T3 mouse fibroblasts, M.dunni fibroblasts, and African green monkey kidney (COS) cells.Transfected cells are assayed for surface expression of the chimericreceptor by FACS to verify that DNA constructs have been successfullyintroduced. Lefranc, (1990) Immunogenetics 32:321-330), followed by atransmembrane domain, followed by amino acids 31-142 of the human Δchain. pIK.F7 contains the transmembrane domain (amino acids 372-395) ofCD4.

To construct this plasmid, the first step was cloning the human IgG3 M1exon (Bensmana and Lefranc, supra). This was done by generating a 0.13kb Bam HI to Bg1II fragment containing the M1 exon by PCR using DNA fromthe human cell line W138 as substrate and oligonucleotides 7 and 8, andcloning it into the Bgl II site of pIK.CD4γ2. The resulting plasmid isreferred to as pIK.CH3/M1-int. Single stranded DNA from this plasmid wasused as template and oligonucleotide 9 was used as the primer to fuseamino acid 445 of human IgG2 to the first amino acid of the IgG3membrane hinge domain by deletion mutagenesis. The fusion is designed togenerate the sequence found at the natural junction between CH3 and M1in membrane-bound IgG molecules. Correctly deleted clones were foundusing oligonucleotide 10 as a probe. The resulting plasmid is referredto as pIK.CD4γ2/M1.

pIK.CD4γ2/M1 was cut with Bgl II and blunted with T4 polymerase, thencut with Nhe I. The resulting 0.83 kb fragment was ligated together withthe 1.3 kb Pvu II to Apa I fragment from pIK.F3 between the Nhe I andApa I sites of pIK.CD4γ2 to generate the plasmid pIK.F7-int. Singlestranded DNA from this plasmid was used as template and oligonucleotide15 was used as the primer to fuse the last amino acid of the IgG3 M1membrane hinge domain to amino acid 372 of CD4 by deletion mutagenesis.Correctly deleted clones were found by using oligonucleotide 16 as aprobe. The resulting plasmid is pIK.F7.

The oligonucleotides used as primers and probes as described above wereas follows:

Oligonucleotides (SEQ ID NO. 10) 2. CGGAGATCTCGTGCGACCGCGAGAGCC (SEQ IDNO. 11) 3. GGAATTCGCTAGCTTTCCAGGAGCGCAAATGTTGTGTC (SEQ ID NO. 12) 4.CGGAGATCTC(A/G)CGCGACCCCGAGAGCC (SEQ ID NO. 13) 7.CGGGATCCAGAGCTGCAACTGGAG (SEQ ID NO. 14) 8. GAAGATCTGACCTTGAAGAAGGTGAC(SEQ ID NO. 15) 9. TCTCCTCCAGTTGCAGCTCCGGAGACAGGGAGAGGC (SEQ ID NO. 16)10. TTGCAGCTCCGGAGAC (SEQ ID NO. 17) 15.CAGCACAATCAGGGCCATGTCCAGCTCCCCGTCCTG (SEQ ID NO. 18) 16.AGGGCCATGTCCAGCT (SEQ ID NO. 19) 17. CGGAATTCGGTACCTCCTGTGCAAGAAC (SEQID NO. 20) 18. CGGAATTCGCCTCCACCAAGGGCCCA (SEQ ID NO. 21) 19.CGGAATTCACGCGTCCCAGTCAGGACACAGC (SEQ ID NO. 22) 20.GAGAGAGATCTGCTAGCGGTCAGGCTGGAACTGAG (SEQ ID NO. 23) 21.GCATGTGTGAGTTTTGTCTGAGGAGACGGTGACCAG (SEQ ID NO. 24) 22.GTTTTGTCTGAGGAGA (SEQ ID NO. 25) 23. GTGACAGTCGACCCCTTGAAGTCCACTTTGGT(SEQ ID NO. 26) 24. CCACCCCTCACTCTGCTTCTC (SEQ ID NO. 27) 25.TCGACCAGCGGCAGCGGCAAGAGCAGCGAGGGTAAGGGTACCA (SEQ ID NO. 28) 26.GATCTGGTACCCTTACCCTCGCTGCTCTTGCCGCTGCCGCTGG (SEQ ID NO. 29) 27.CTCCTGTAGTAGCACCTGACCCTTACCCTCGCTGCT (SEQ ID NO. 30) 28.AGCACCTGACCCTTACConstruction of pIK.F15neo:

To direct the expression of a fusion protein consisting of the K/L/H SAbform of mAb 98.6 linked at amino acid 445 of the IgG1 heavy chain to the18 amino acid IgG3 M1 membrane hinge, which was in turn fused to the CD4transmembrane domain (amino acids 372-395) and Δ cytoplasmic domain(amino acids 31-142), pIK.F15neo was constructed by inserting the 1.5 kbNsi I fragment of pIK.98.6-K/L/H between the Nsi I sites of pIK.F7neoand a clone of the correct orientation was selected.

Production of Retrovirus in Mammalian Cells

Single or double genome KAT packaging plasmids, for examplepIK6.1MMSVampac,pIK6.1MCVampac, or pIk6.1amenvATG and pIK6.1gagpolATG(all described above), together with KAT retroviral constructs, forexample, but not limited to pRTD2.2F3, pRTD2.2SVGF3, pRTD2.2SSAF3,pRTD2.2SVGF3E-, pIKT2.2SVGF3, pRTD2.2F15 (as described above), preparedas described above, are introduced into mammalian cells that can producevirus by standard means such as calcium phosphate cotransfection (Wigleret al., Proc. Natl. Acad. Sci. USA 76:1373-1377 (1979)). Mammalian cellsthat can produce virus and that may be transfected by the KAT constructsof the invention include, but are not limited to, human embryonic kidneycells such as 293 cells, tsa201 cells, mouse 3T3 mouse fibroblasts, M.dunni fibroblasts, and African green monkey kidney (COS) cells.Transfected cells are assayed for surface expression of the chimericreceptor by FACS to verify that DNA constructs have been successfullyintroduced.

Viral supernatants are harvested using standard techniques such asfiltration of supernatants 48 hours post transfection. The viral titeris determined by infection of 10⁶ NIH 3T3 cells with an appropriateamount of viral supernatant, in the presence of 8 μg/ml polybrene (SigmaChemical Co., St. Louis, Mo.). 48 hours later, the transductionefficiency of the 3T3 cells is assayed by both FACS analysis andSouthern blotting.

High Efficiency Transduction of Target Cells

In the method of the invention the KAT constructs of the invention arefurther used to transduce mammalian target cells with a foreign gene athigh efficiency by cocultivation of KAT transfected cells with themammalian target cells. In a preferred embodiment, desired virusproducing cells, such as 293 cells, are transfected with the appropriateKAT constructs, then 24 hours post transfection, the transfected 293cells are cocultivated for 48 hours with the purified mammalian targetcells, such as CD8+ T cells. Alternatively, fresh media is added 24hours post-transfection. Forty-eight hours post-transfection, virussupernatants are harvested, filtered through a 0.45 μ filter and used toinfect target cells. The target cells are harvested using standardprocedures, expanded and tested for transduction efficiency, bywell-known techniques such as flow cytometry or Fluorescence-activatedCell Sorter (FACS) analysis and Southern blot DNA analyses. Transductionefficiency is defined as the percentage of positive transduced cells asmeasured by FACS or Southern blot analysis compared to controls.

Using the KAT constructs transfected into human 293 cells to producevirus, a from 5 to 50-fold increase in viral titer as determined bysupernatant infection of established cell lines, such as 3T3, isobtained, when compared to virus produced by the previously describedCOS transient virus production system (Landau and Litman, supra). Inaddition, primary human cells such as hematopoietic stem cells and humanT cells, are transduced at levels 3 to 20 fold greater by cocultivationwith KAT plasmid transfected 293 cells, than traditional packaging linessuch as PA317 (Miller and Buttimore, supra).

While not wishing to be bound by any particular theory of the invention,it is believed that the high efficiency transduction of human targetcells obtained using the cocultivation transduction method of theinvention is mediated by cell-cell contact of the retrovirally infectedhuman 293 cells with the target cells. The component of human 293 cellswhich effects high efficiency transduction of various target cells isexpected to be a protein or lipid synthesized by the 293 cells. Todetermine the active component of this system, the membrane proteins andlipids of 293 cells are purified using known procedures and the abilityof various purified components is tested for its ability to effect thetransduction efficiency of the target cells. Once the active componentis identified it can be synthesized by recombinant DNA or chemicaltechnique. These synthesized components may be incorporated into virusparticles to enhance the transduction efficiency of supernatants.

Suitable target cells are any mammalian cells of interest, and include,but are not limited to lymphocytes, particularly cytotoxic T cells,human hematopoietic stem cells, fibroblasts, epithelial cells,endothelial cells, myoblasts, retinal epithelial cells, islets ofLangerhans, adrenal medulla cells, osteoblasts, osteoclasts, neurons,glial cells, ganglion cells, embryonic stem cells, and hepatocytes.

The genes which may be introduced into the target cells include, but arenot limited to genes encoding chimeric receptors for signal transductionin lymphocytes, such as those described in copending U.S. patentapplication Ser. No. 988,194, filed Dec. 9, 1992, the disclosure ofwhich is incorporated in its entirety herein by reference; growthfactors, such as G-, M- and GM-colony stimulating factor (CSF),epidermal growth factor, platelet derived growth factor, transforminggrowth factor (TGF) and stem cell growth factor (SCF); lymphokines suchas the interleukins; hormones such as ACTH, somatomedin, insulin,angiotensin; and coagulation factors, such as Factor VIII and Factor IX;the Multidrug Resistance Drug (MDR) gene; human adenosine deaminase(ADA); glucose cerebrosidase; the normal β-globin gene and erythopoietin(EPO).

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of thedisclosure or the protection granted by Letters Patent hereon.

EXAMPLE I Transient Production of High Titer Recombinant Retrovirus

Cell Growth, Transfection and Infection of Established Cell Lines

Human embryonic kidney cells, designated 293 cells (ATCC CRL 1573, ATCC,Rockville, Md.) cells were grown in DMEM (JHR Biosciences, Lenexa,Kans.), 1 g/l glucose, 10% Donor calf serum (Tissue Culture Biologics,Tulare, Calif.) and split 1:10 every 3 days. 3T3 (ATCC CRL1573) cellswere grown in DMEM (JHR Biosciences), 4.5 g/l glucose, 10% Donor calfserum (Tissue Culture Biologics) and split 1:10 every 3 days. COS (ATCCCRL1650) cells were grown in DME/F12 (GIBCO, Grand Island, N.Y.), 10%fetal bovine serum (Tissue Culture Biologics, Tulare, Calif.) and split1:10 every 3 days. tsa201 cells, a derivative of 293s which contain thetemperature sensitive mutant of the SV40 T antigen co-transfected withthe neomycin resistance gene (Heinzel et al., J. Virol. 62 (10):3738-3746 (1988)), were grown in DME/F12 (GIBCO), 10% fetal bovine serum(Tissue Culture Biologics) and split 1:10 every 3 days. 293 cells andtsa201 cells were plated 1×10⁶ and 0.5×10⁶ cells per 10 cm plate,respectively, 48 hours prior to transfection. COS and 3T3 cells wereplated at 0.5×10⁶ cells per 10 cm plate 24 hours prior to transfection.10 μg of each plasmid, alone or in various combinations, was transfectedby calcium phosphate coprecipitation (Wigler at al., supra) for all celltypes. 24 hours following transfection, the media was changed. 24 hourslater, viral supernatants were harvested and filtered through a 0.45 μmfilter and flash frozen on dry ice. 3T3 cells were plated at 0.5×10⁶cells per 10 cm plate 24 hours prior to infection. Infections werecarried out in 5 ml of media containing viral supernatant and 8 μg/mlpolybrene (Sigma Chemical Co., St. Louis, Mo.). 24 hours followinginfection, the media was changed to polybrene-free media and the cellswere grown for an additional 24 hours.

293 Cells Produced High Titer Retrovirus Following TransientTransfection

293 cells were assayed for their ability to transiently producerecombinant retrovirus upon cotransfection with the either the KATpackaging plasmid(s) pIK6.1MCVampac or pIK6.1amenvATG andpIK6.1gagpolATG,and the retroviral vectors pRTD2.2F3, pRTD2.2SVGF3,pRTD2.2SSAF3, pRTD2.2SVGF3E-, pIKT2.2SVGF3, and pRTD2.2F15, encoding theF3 or F15 chimeric receptors, by harvesting viral supernatants 48 hourspost transfection, followed by infection of mouse 3T3 cells, and FACsanalysis 48 hours later.

For FACS analysis, infected 3T3 cells are removed from the culture dishin the absence of trypsin and are processed for FACS analysis afterincubation in 40 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA. Cells arewashed 1× with phosphate buffered saline (PBS) plus 2% (FCS) fetal calfserum (Hyclone), followed by incubation with the appropriateFITC-conjugated detection antibody in the presence of PBS plus 2% FCS ata density of 1×10⁶/ml for 30 minutes at 40° C. The cells are washed 3×with PBS plus 2% FCS, and finally resuspended in 0.5 ml PBS and analyzedby flow cytometry.

The results of FACS analysis are shown in FIG. 2. 293 cellscotransfected pIK6.1ampac and pRTD2.2F3 express high levels of F3 ontheir surface (FIG. 2B), compared to mock (control) transfected cells(FIG. 2A). 3T3 cells infected with viral supernatants harvested fromtransfected 293 cells revealed two well separated peaks corresponding touninfected and infected 3T3 cells (FIG. 2D), which was significantlydifferent compared to the FACS profile of transfected 293 cells (FIG.2B) or mock infected 3T3 cells (FIG. 2C).

Table 1 demonstrates that cotransfection of KAT packaging plasmids andKAT retroviral constructs results in the production of high titer viralsupernatants 48 hours following transfection, as assayed by 3T3infection and FACS analysis. Cotransfection of pIK6.1ampac and pRTD2.2F3yields viral supernatants that transduce 50% of the 10⁶ 3T3 cellsinitially present at the time of infection. In contrast, virus producedby transient cotransfection in COS cells, as described by Landau andLitman (Landau and Litman, supra) was 10-fold less than the titersdescribed by cotransfection of KAT plasmids into 293 cells. Virusproduction is highly reproducible in four transfection experiments,where duplicate 3T3 infections were carried out. In contrast, nodetectable 3T3 infection is observed following transfection of theretroviral construct pRTD2.2F3 alone, demonstrating that viralproduction is dependant upon the presence of the packaging construct andthe retroviral vector. High titer virus production is also dependantupon the presence of the retroviral construct. Transfection of pIKF3expression vector alone, or cotransfection of pIKF3 expression vectorand pIK6.1MMSVampac yields supernatants that fail to transduce 3T3cells.

TABLE 1 % % 3T3 Construct Packaging Function Transfection TransductionpRTD2.2F3 — 52 0/0 pRTD2.2F3 — 55 0/0 pRTD2.2F3 pIK6.1MCVampac 80 49/50pRTD2.2F3 pIK6.1MCVampac 85 50/49 pRTD2.2F3 pIK6.1MCVampac 83 47/43pRTD2.2F3 pIK6.1MCVampac 85 49/48 pRTD2.2F3 pIK6.1gagpo1ATG, 78 27/77pIK6.1amenvATG pRTD2.2F3 pIK6.1gagpolATG, 78 25/26 pIK6.1amenvATG pIKF3— 67 0/0 pIKF3 — 59 0/0 pIKF3 pIK6.1MCVampac 90 0/0 pIKF3 pIK6.1MCVampac90 0/0 pRTD2.2ssaF3 pIK6.1MCVampac 78 33/35 pRTD2.2svgF3 pIK6.1MCVampac84 44/39 pRTD2.2svge-F3 pIK6.1MCVampac 81 42/43 pRTD2.2F15pIK6.1MCVampac 93 70/70 pRTD2.2F15 pIK6.1MCVampac 91 69/70

High titer virus can also be produced by cotransfection ofpIK6.1amenvATG, pIK6.1gagpolATG and pRTD2.2F3 (Table 1). Although thetransfection efficiency of the later plasmids was approximately equal tothe transfection efficiency of pIK6.1MCVampac and pRTD2.2F3, virusproduction was reduced by a factor of 2 to 27%. Similar results havebeen described by Landau and Litman (Landau and Litman, supra), wherethey observed a 5-fold decrease. The overall efficiency of the KATsystem, using one or two genome packaging plasmids, is still 10 to20-fold greater then that described for the COS cell system.

The high 3T3 cell transduction efficiency observed by FACS analysis ofviral supernatants produced following KAT plasmid transfection of 293cells was confirmed by Southern blotting of integrated proviral DNA frominfected 3T3 cells. High molecular weight DNA was prepared 48 hours postinfection and digestion of 10 μg of DNA with Eco RV. The samples wereelectrophoresed on a 0.8% agarose gel, transferred to Zetabind andprobed with a 605 base pair fragment encoding the zeta transmembrane andcytoplasmic domains. Eco RV digestion of the transfected plasmidpRTD2.2F3 yielded a 4.2 kb band. Eco RV digestion of pRTD1.2F3, whichcontains MMLV 5′ and 3′ LTRs, yielded a 3.6 kb fragment. Following virusinfection, integration and duplication of the 3′ LTR, Eco RV digestionshould yield a 3.6 kb fragment. This allows determination of thepresence of integrated proviral DNA in the target cells. Table 2 givesthe sizes of the expected bands from transfected plasmid DNA andintegrated provirus following Eco RV digestion and hybridization to thezeta probe.

TABLE 2 EcoRV Fragment Size (in Kb) Hybridizing to Δ Probe RetroviralConstruct Transfected Plasmid Integrated Provirus pRT.D 2.2F3 4.20 3.60pRT.D 2.2SSAF3 3.80 3.20 pRT.D 2.2SVGF3 4.17 3.57 pRT.D 2.2SVGE-F3 4.223.61 pRT.D 2.2F15 4.47 3.87

Genomic DNA prepared from infected 3T3s was digested with Eco RV and 10μg of digested DNA from infected and control cells were electrophoresedon a 0.8% agarose gel, transferred to Zetabind and probed with a 605base pair fragment encoding the Δ transmembrane and cytoplasmic domains.Only the DNA derived from 3T3 cells infected with supernatants obtainedfollowing cotransfection of 293 cells with pRTD2.2F3 and pIKMCVampacyielded a 3.6 kb fragment (FIG. 3A, lanes 4 and 5), identical to thefragment seen in the Eco RV digested pRTD1.2F3 plasmid control lanes(FIG. 3A, lanes 11-14), indicative of integrated provirus. Quantitationof southern blots by scanning densitometry and comparison to plasmidstandards representing 0.1 to 3.0 copies, in 3-fold increasingincrements (FIG. 3A, lanes 11-14), was consistent with a transductionefficiency of with a transduction efficiency of 0.5 copies/cell/ml ofviral supernatant. The transduction efficiency was identical to theefficiency observed by FACS analysis. The probe did not detect a band inDNA from 3T3 cells infected with supernatants derived from mocktransfected 293 cells (lane 1), 293 cells transfected with pRTD2.2F3alone (FIG. 3A, lanes 2 and 3), transfected with the expression vectorpIKF3 alone (FIG. 3A, lanes 6 and 7) or cotransfected withpIK6.1MCVampac and pIKF3 (FIG. 3A, lanes 8 and 9), which is alsoconsistent with the FACS analysis.

Three additional retroviral constructs, two which differed in the viralbackbone, pRTD2.2SSAF3 (FIG. 3B, lane 4), pRTD2.2SVGF3 (FIG. 3B, lane5), pRTD2.2SVGE-F3 (FIG. 3B, lane 6), and one which differed in thechimeric receptor insert, pRTD2.2F15 (FIG. 3B, lanes 7 and 8), werecotransfected into 293 cells with pIK6.1MCVampac, the supernatant usedto infect 3T3 cells, followed by both FACS analysis (Table 1) andsouthern blotting (FIG. 3B). All of the F3 constructs showed similartiter by both FACS analysis (Table 1) and hybridized to the zeta probewith similar intensities, as expected. The F15 retrovirus hadapproximately 50% greater titer as determined by FACS analysis (Table1), as well as by densitometric analysis of the Southern blots.Retrovirus as produced in 293 with each of the vectors, upon infection,yielded the correct size for the integrated provirus. Therefore, theFACS and Southern blotting results from 5 KAT retroviral constructsdemonstrate that high titer retrovirus can be produced in 293 cells,that production was dependent upon cotransfection of the retroviralconstruct and packaging functions, and production of high titerretroviral supernatants in 293 cells does not lead to any unusualrearrangements of the retroviral constructs.

Virus Production in Mammalian Cell Lines:

Seven additional cell lines were screened for their ability to produceretrovirus by cotransfection with KAT plasmids, followed by virusharvest and 3T3 infection (Table 3).

TABLE 3 Packaging Surface Retro-F3 Cell Type Construct CD4% 3T3 inf %Constr. 293 Mock 1 0/0 Mock 293 pIK6.1MCVampac 88 39/38 pRTD2.2-F3 293pIK6.1MCVampac 88 41/38 pRTD2.2-F3 COS Mock 0 ND Mock COS pIK61MCVampac58  12/14. pRTD2.2-F3 COS pIK6.1MCVampac 58 14/15 pRTD2.2-F3 143B Mock 0ND Mock 143B pIK6.1MCVampac 54  1/1. pRTD2.2-F3 143B pIK6.1MCVampac 50 1/1. pRTD2.2-F3 HELA Mock 0 ND Mock HELA pIK6.1MCVampac 48 0/0pRTD2.2-F3 HELA pIK6.1MCVampac 54 0/0 pRTD2.2-F3 L929 Mock 0 ND MockL929 pIK6.1MCVampac 1 0/0 pRTD2.2-F3 L929 pIK6.1MCVampac 1 0/0pRTD2.2-F3 3T3 Mock 0 0/0 Mock 3T3 pIK6.1MCVampac 39  2/3. pRTD2.2-F33T3 pIK6.1MCVampac 44  4/3. pRTD2.2-F3 CHO D- pIK6.1MCVampac 0 0/0pRTD2.2-F3 CHO D- pIK6.1MCVampac 0 0/0 pRTD2.2-F3

CD4 surface expression and virus production was absent from L929 and CHOD- following cotransfection of pIK6.1MCVampac with pRTD2.2F3. However,these cell lines were highly transfectable under conditions with aplasmid encoding the lac z gene was used. FACS analysis of transfectedHELA, 143B, 3T3 and COS demonstrated high surface CD4 expression, with atransfection efficiency of approximately 50% for all four cell types.However, virus production among these cells was substantially different.HELA and 143B cells produced no virus at all, whereas 3T3 cells producedvirus capable of 3% 3T3 transduction/ml of frozen supernatant.Cotransfection of COS cells with KAT plasmids, even in the absence ofDNA replication of the retroviral construct, produced virus with titersof 4.5-fold greater than that produced by 3T3 cells. These titers,without plasmid replication of the viral vector construct, are 200 foldgreater than those described by Landau and Litman (Landau and Litman,supra). This demonstrates that the KAT constructs are unique in theirability to produce retrovirus upon transfection of a wide variety ofcells, without plasmid replication. Given the 100 fold increase thatLandau and Litman observed with plasmid replication of the viral vectorconstruct, transfection of KAT packaging function and retroviral vectorplasmids that support plasmid replication, into hosts that supportplasmid replication, could potentially further increase titer 10 to 100fold and further increase the utility of KAT transfected cells to infectcell types that are currently difficult to infect.

EXAMPLE II High Efficiency Transduction of Human T Cells

This example demonstrates the method of the invention in which 293 cellstransfected with the KAT constructs are able to transduce primary, humantarget CD8+ T cells by cocultivation with high efficiency.

Construction of Retroviral Vectors and Packaging Plasmids

KAT constructs were prepared as described above in Example I.

Isolation and Activation of Human CD8+ T Cells from Peripheral Blood

Primary human CD8+ T cells were purified from the peripheral blood ofhealthy donors as follows: Peripheral blood mononucleocytes (PBMCs) wereisolated from human blood by Ficoll-Hypaque density gradientcentrifugation. PBMCs were washed three times with D-PBSE/CMF (PBScontaining 1 mM EDTA, Ca and Mg free), resuspended at 5×10⁷ cells in 4ml of D-PBSE/CMF containing 0.5% of human gamma globulins, and incubatedat room temperature for at least 15 minutes. After incubation, CD8+ Tcells were purified from the PBMC cell suspension by positive panning.Specifically, the PBMC suspension was loaded into a pre-washed T-25tissue culture flask coated with an antibody specific for the human CD8receptor (AIS CD8 selection flask (Applied Immune Sciences, Santa Clara,Calif.)) at a density of 5×10⁷ cells per 4 ml per T-25 flask. Cells wereincubated for one hour at room temperature, and the non-adherent cellsremoved by gentle pipetting and washing the flask three times with theD-PBSE/CMF. The CD8+ T cells were simultaneously released from the flaskand activated by adding 10 ml of T cell medium (see below forcomposition) containing 10 ng/ml OKT3 (Ortho Pharmaceuticals, Raritan,N. J.) and 10% IL2 (Pharmacia). Cells were incubated with this media for48 hours, harvested from the flask, and washed once with T cell medium,and finally resuspended in fresh T cell medium plus 10% IL2 at a densityof 0.5-1.0×10⁶/ml in 24 well plates.

In order to remove residual cells (usually present at 2-3%) whichcross-reacted with either the CD4-specific antibody used for detectionof F3 surface expression, or the human Fc-specific antibody used todetect F15 surface expression, the enriched CD8+ T cell population wassubjected to a further round of purification in which the contaminatingcells were removed by negative panning, using AIS selection flasksdescribed above, coated with either the anti-CD4 or anti-human Fcantibody. Specifically, the enriched CD8+ T cell population wasincubated in the selection flask for one hour, and then non-adherent(i.e., highly purified CD8+ T cells) were removed. Cells weresubsequently washed, and allowed to recover for 24 hours in the T cellmedium plus 10% IL2 for 24 hours. CD8+ T cells prepared in this mannerwere greater than 95% CD8+ and CD3+, and less than 0.5% CD4+ or FC+, andwere subsequently employed as targets for retroviral transduction.

Retroviral Transduction of CD8+ T Cells by Cocultivation or SupernatantInfection:

293 cells were plated at 1×10⁶ cells/6 well plate, and then transfectedwith the appropriate construct after 48 hours as described above. 24hours post transfection, the transfection media was removed and replacedwith T cell growth media (see below for composition).

(a) Cocultivation: 2 to 4 hours later, 0.5×10⁶ purified and activatedhuman CD8+ T cells prepared as described above (usually at day 4 or 5post-purification/activation) were added per well containing thetransfected 293 cells, and polybrene added at a final concentration of 2μg/ml. 24 hours after plating the 293 cells for the initialtransfection, a second set of 293 cells were plated and transfected asdescribed above. 24 hours after the initial cocultivation, T cells wereremoved from the first cocultivation and transferred to the second 293transfection plate for an additional 24 hours of cocultivation employedthe same conditions. Similar conditions were employed for transductionof CD8+ T cells by cocultivation with either transiently transfected 3T3cells, or the stable PA317 producer cell line 40.39 (see below).

(b)Supernatant infection: 0.5×10⁶ purified and activated human CD8+ Tcells prepared as described above (usually at day 4 or 5post-purification/activation) were incubated with 1 ml of fresh T cellmedium (plus 10% IL2 and 2 μg/ml polybrene) together with 1 ml of viralsupernatant obtained from the 293 transient transfection systemdescribed above, or from the stable PA317 producer cell line 40.39 (seebelow). After an 8 hour incubation period, 1.5 ml of medium was removedfrom each well, and replaced with 0.5 ml of fresh T cell medium togetherwith 1.0 ml of viral supernatant (polybrene at 2 μg/ml and IL2 at 10%).After a 12 hour incubation period, the two step supernatant procedurewas repeated.

For both cocultivation and supernatant infection, CD8+ T cells wereallowed to recover for a 24-28 hour period in fresh T cell medium plus10% IL2. Cells were then analyzed by flow cytometry for surfaceexpression of either CD4 (for the CD4-ΔF3 receptor) or Fc for the F15antibody-Δ receptor) in order to determine transduction efficiencies. Tcells which were under cocultivation with transfected 293 cells weregently removed as a suspension from the 293 monolayer. Both cocultivatedand supernatant infected T cells were washed 1× with phosphate bufferedsaline (PBS) plus 2% (FCS) fetal calf serum (Hyclone). T cells were thenincubated with the appropriate FITC-conjugated detection antibody in thepresence of PBS plus 2% FCS at a density of 1×10⁶/ml for 30 minutes at40° C., washed 3× with PBS plus 2% FCS, and finally resuspended in 0.5ml PBS and analyzed by flow cytometry.

The transduced CD8+ T cell population was subsequently maintained in Tcell medium (10% FCS, Hyclone; RPMI1640, CellGro; 10 mM Hepes buffer(Gibco); 1% Sodium pyruvate (Gibco); 1% non-essential amino acids(Gibco); 2 mM glutamine (Gibco); 25 μM 2-mercaptoethanol (Sigma) and 1%streptomycin/penicillin). T cells were periodically re-stimulated every7 to 10 days by the addition of OKT3 at 10 ng/ml or by exposing thecells to immobilized OKT3 in a T-25 tissue culture flask at a density of1-2×10⁷ CD8+ T cells/10 ml T cell medium plus 10% IL2. Cells wereincubated for 48 hours, washed 1× with T cell medium, and resuspended infresh medium plus 10% IL2 at 0.5-1.0×10⁶/ml.

Analysis of CD8+ T cell Transduction:

Transduction efficiency of primary human CD8+ T cells by retrovirusproduced transiently using the KAT system was compared to retrovirusproduced from a high-titer, stable producer clone derived from theamphotropic packaging line PA317 (Miller and Buttimore, supra). Thestable producer clone 40.39, which transduces the F3 chimeric receptorwas isolated by transfection of the ecotropic packaging line gpe(Markowitz et al.supra) with pRTD4.2F3, followed by supernatant harvest48 hours post transfection and infection PA317 in the presence of 8micrograms/ml of polybrene (Miller and Buttimore, supra). Individualclones were obtained by limiting dilution and 50 were screened for virusproduction by isolation of viral mRNA from the media of clones, followedby dot blot hybridization using a 603 base pair zeta chain probe. Theclone that gave the strongest hybridization signal, clone 40.39, wasassayed by limiting dilution infection of 10⁶ NIH 3T3 cells followed byflow cytometry. 50 μl of supernatant transduced 17% cells, equivalent to340% or an average of 3.4 proviral copies/cell/ml. The transductionefficiency following a 48 hour cocultivation with primary human CD8+ Tcells with 40.39 producer cells was 1%-3% CD4+ (Table 4).

This result was compared to the transduction efficiency following theKAT transient-transfection and cocultivation method of the invention,which was used to transduce the chimeric receptor F3 and F15 into CD8+ Tcells (FIG. 4). Four experiments were carried out in which CD8+ T cellswere cocultivated on transfected 293 cells for 48 hours, followed byharvest and growth of T cells for 14 days and analysis of transductionefficiency by FACS as described above. The transduction efficiency ofCD8 cells with both F3 and F15 constructs varies between 8% and 38%, andappears to be highly donor dependent. However, on average, thisefficiency is 8 to 12-fold greater than the transduction efficiencyobtained by cocultivation with the high-titer stable PA317 clonestested. In addition, the high transduction efficiency is not specific toF3 constructs because F15 constructs are transduced at similarefficiencies (FIG. 4). This data demonstrates that CD8 T cells can betransduced at efficiencies that are at least 5 fold greater than orequal to any other published reports, and that generation of stableproducers are not required.

Supernatants from transduced T cells, 3 weeks post-transduction, weretested in an extended S+L-assay (Miller et al., Mol. Cell. Biol.5:431-437 (1985)) and shown to be free of replication-competentretrovirus.

High Efficiency Transduction is Mediated by Cell-cell Contact

In order to explore the mechanism of the high efficiency CD8 T celltransduction following transient transfection of KAT plasmids andcocultivation with CD8+ T cells, the transduction efficiency of CD8+ Tcells using the following approaches was compared: (1)infection withsupernatants derived from a high titer, stable PA317 producer line,(2)cocultivation with a high titer, stable PA317 producerline(3)infection with supernatants derived from transient transfectionof NIH 3T3 cells with pIK 6.1MMSVampac and pRTD4.2F3 (4)48 hourcocultivation with NIH 3T3 cells following transient transfection withpIK6.1MMSVampac and pRTD4.2F3 (5)infection with supernatants derivedfrom transient transfection of 293 cells with pIK6.1MCVampac andpRTD2.2F3 and (6) 48 hour cocultivation with 293 cells followingtransient transfection with pIK6.1MCVampac and pRTD2.2F3 (Table 4). Foreach transient transfection experiment, duplicate plates of transfectedcells were used to harvest media for supernatant infection of 3T3 cellsand duplicate plates were used for cocultivation of CD8 T cells. Thesame approach was used for stable producers.

TABLE 4 % Virus 3T3 titer T-cell Pkg. Production Infection Super- Trans-Expt.# Line Method Method natant duction 1A PA317 PA317, StableSupernatant 70% 1 1B PA317 PA317, Stable co-cultivation ND¹ 3 1C 3T3KAT, Transient co-cultivation 26% 3 1D 293 KAT, Transient co-cultivation14% 10 2A PA317 PA317, Stable Supernatant 30% 1 2B PA317 PA317, Stableco-cultivation 30% 1 2C 293 KAT, Transient Supernatant 45% 1 2D 293 KAT,Transient co-cultivation 45% 14 ND¹ = not determined

Supernatant infection of CD8+ T cells was 1%, whether the virus wasproduced in 293 cells, 3T3 cells or a stable PA317 producer (Table 4,experiments 1A, 2A and 2C). In contrast, cocultivation of CD8 T cellswith 293 cells cotransfected with pIK6.1MCVampac and pRTD2.2F3, resultedin 10% to 14% CD8 T cell transduction (Table 4, experiment 1D, 2D), 10to 14-fold greater than all supernatant infections, includingsupernatants produced by cotransfection of these plasmids into 293cells. This demonstrates that cell-cell contact is responsible for highefficiency transduction of CD8+ T cells. In addition, the efficiency ofKAT transfection followed by cocultivation is 1 to 3-fold greater thanthe transduction efficiency of cocultivation with a stable PA317producer when 3T3 cells are used (compare 1B and 2B with 1C, table 4)and 5-10 fold greater when 293 cells are used. This data confirms that293 cells have unique properties that support high efficiencytransduction of mammalian cells.

While not wishing to be limited to any particular theory of theinvention, these results suggest that high titer virus production intothe culture media is not sufficient for efficient T cell transductionand that the high efficiency transduction observed is mediated bycell-cell contact of 293 cells and CD8+ T cells, resulting in up toten-fold greater efficiencies.

The results presented in this example demonstrate that, in the absenceof selection, 10-40% of the CD8+ T cells were virally transduced, asignificantly greater transduction frequency compared to prior results.

EXAMPLE III Transduction of Primary Human Hematopoietic Stem Cells

This example describes the use of the KAT constructs and method of theinvention to transduce primary human CD34+ bone marrow stem cells.

Preparation of Bone Marrow Cells

Human bone marrow was obtained from healthy volunteers. It was firstfractionated into a mononuclear cell fraction over a Ficoll gradient(Pharmacia, Piscataway, N. J.). The CD34+ cells are isolated usingpositive selection on a CellPro CEPTRATE LC™ affinity column (CellPro,Bothell, Wash.). Post-purification FACS analysis provided a populationof approximately 90% CD34+ cells. This population of cells was thenplated in 24 well plates at a density of 5×10⁵ cells/ml in Myeloid LongTerm Culture Medium supplied as a complete medium from Terry Fox Labs,(Vancouver, Canada) in the presence of 100 ng/ml human Stem Cell Factor(hSCF) (R&D Systems, Minneapolis, Minn.) 50 ng/ml hIL-3, and 10 ng/mlhIL-6 for 48 hours.

Transduction of CD34+ Bone Marrow Stem Cells

293 cells were transfected by first plating at a density of 1×10⁶cells/6 well plate 48 hours prior to transfection, followed bytransfection with 10 μg each of pRTD2.2F3 and pIK6.1MCVampac.Twenty-four hours later, transfection media was removed, replaced with Tcell growth media, as described in Example II, plus 50 ng/ml hIL-3, 100ng/ml hSCF, and 10 ng/ml hIL-6. Two to four hours later, the transfected293 cells were cocultivated with 5×10⁵ purified CD34+ cells/well in thepresence of 8 μg/ml polybrene. After 48 hours, the cells were collectedoff of the 293 monolayer, and replated in Myeloid Long Term Media withgrowth factors as described above. Cultures were replenished with mediaplus growth factors daily via demi-depopulation. Four days later, themedia was replenished and G-CSF was added at 2 ng/ml plus 20 ng/ml hSCFto promote differentiation into granulocytes. Four to six days later,cells were analyzed for surface expression of human CD4 from thetransduced gene and CD15, a granulocyte marker. In addition, DNA wasprepared for Southern blot analysis.

FIG. 5 shows the FACS analysis of the transduced hematopoieticstem/progenitor cells after 14 days of growth and differentiation intogranulocytes. Panel A shows the forward and side scatter gates used inthe analysis of all cell populations in the Figure. In panel B are shownthe untransduced cells stained with the isotype control antibodies (FITCand PE). In panel C are shown the untransduced cells stained withantibodies for human CD4 (transduced gene, y axis) and CD15 (granulocytedifferentiation marker, x axis). In panel D, KAT packaging system wasused in conjunction with 293 cell co-cultivation to transduce thehematopoietic stem/progenitor cells. A comparison of the top rightquadrant for panels C and D indicate that 5-6% of the transduced cellsexpressed the CD4 protein.

Southern Blot Analysis of Transduction Efficiency

Southern blot analysis was carried out to determine whether thehematopoietic stem/progenitor cells were infected by retrovirus producedwith the KAT system. Genomic DNA was prepared from differentiated stemcells and digested with Eco RV. 10 μg of DNA from infected (FIG. 6, lane2) and control cells (FIG. 6, lane 1), as well as Eco RV-digestedplasmid DNA equivalent to 0.12, 0.6, 1.2 and 6.0 copies per diploidgenome of pRTD1.2F3 (FIG. 6, lanes 4-7) and 5 copies per diploid genomeof pRTD2.2F3 (FIG. 6, lane 8) were electrophoresed on a 0.8% agarosegel, transferred to Zetabind and probed with a 605 base pair fragmentencoding the zeta transmembrane and cytoplasmic domains. Eco RVdigestion of the transfected plasmid pRTD2.2 yields a 4.2 kb band (FIG.6, lane 8). Eco RV digestion of pRTD1.2, which contains MMLV 5′ and 3′LTRs, yields a 3.6 kb fragment (FIG. 6, lanes 4-7). Following virusinfection, integration and duplication of the 3′ LTR, Eco RV digestionshould yield a 3.6 kb fragment. In infected CD34+ cells, the probehybridized to the appropriate 3.6 kb band, corresponding to integratedprovirus (FIG. 6, lane 7). Control cells lacked a proviral band, howeverthe probe hybridized to bands that corresponded to the endogenous zetagene sequences (FIG. 6, lane 8). Scanning densitometry was used toquantitate transduction efficiency and demonstrated that the averageproviral copy number per cell in infected cells was 0.5 (50%transduction). In addition, densitometry of the endogenous bandsconfirmed that equal amounts of DNA were loaded in the lanescorresponding to infected and uninfected cells.

In a second experiment, the transduction efficiency of a high titerPA317 producer clone was compared to the transduction efficiency ofvirus produced by the KAT system. 293 cells were transientcotransfection with pIK6.1MCVampac and pRTD2.2F3,isolation of CD34+cells, cocultivation, purification of infected cells was carried out asdescribed above. Clone 40.39, described above in Example II, was platedat 5×10⁵ cells/6 well plate 24 hours prior to initiation ofcocultivation with CD34+ cells. Isolation of CD34+ cells, cocultivation,purification of infected cells was carried out as described for 293cells. Transduction efficiency was analyzed by southern blotting of EcoRV digested DNA as described above and is shown in FIG. 7. The bandpresent in DNA isolated from CD34+ cells cocultivated with KAT plasmidshybridized to a 3.6 kb band (FIG. 7, lane 2), identical in size to EcoRV digested plasmid DNA (FIG. 7, lanes 4-7) and corresponding tointegrated provirus. Hybridizing bands were absent from DNA isolatedfrom CD34+ cells cocultivated with either mock transfected 293 (FIG. 7,lane 1) cells or 40.39 cells (FIG. 7,lane 3). The plasmid standardsranged from 0.3 to 10 copies of integrated provirus per cell. Therefore,the absence of a band in the PA317 lane suggests that KAT transductionis at least 10-fold more efficient.

FIG. 8 shows the results of FACS analysis of the transducedhematopoietic stem/progenitor cells after 18 days of growth anddifferentiation into granulocytes. Panel A shows the forward and sidescatter gates used in the analysis of all cell populations in theFigure. In panel B are shown the untransduced cells stained with theisotype control antibodies (FITC and PE). In panel C are shown theuntransduced cells stained with antibodies for human CD4 (transducedgene, y axis) and CD15 (granulocyte differentiation marker, x axis). Inpanel D a PA317 clone (78.81), with a titer equivalent to 107 neor CFUclones/ml on 3T3 plates, was used as a stable viral producer in aco-cultivation with the hematopoietic stem cells. In panel E, KATpackaging retroviral vectors were used in conjunction with 293 cellco-cultivation to transduce the hematopoietic stem/progenitor cells. Acomparison of the top right quadrant for panels C and D, and C and Eindicates that 1.7% of the PA317 transduced cells expressed the CD4protein as compared to 24% of those transduced using the KAT constructs.

EXAMPLE V Production of a Single Vector 293 or tsa54 Stable PackagingClone

This example describes the production and use of a single vectorpackaging clone. Human 293 or tsa54 cells were plated at 5×10⁵ per 10 cmplate in DME (JRH . Biosciences, Lenexa, Kans.), 1 g/l glucose, 10%Donor calf serum (Tissue Culture Biologics, Tulare, Calif.) 48 hoursprior to transfection. 10 μg MCV ampac UTΔ and 0.1 μg MC1 neo (Thomasand Capecchi Cell 51:503-512 (1987)) were cotransfected by calciumphosphate precipitation (Wigler et al. Cell 16:777 (1979)). Clones couldwere also generated as efficiently by co-electroporation of the vectors(Shigekawa and Dower Biotechniques 6 (8):742-751 (1988)). 18 hourspost-transfection the media was changed. 24 hrs. later the cells weresplit to duplicate plates of 1:10, 1:20 and 1:50 in media plus 1 mg/mlG418 (Geneticin, GIBCO, Grand Island, N.Y.). Media was changed every 3days for 14 days. Clones were picked to 24 well plates and grown toconfluence. Media was collected from wells, filtered through a 0.45 μmfilter and flash frozen on dry ice. Cells were resuspended in media plus10% DMSO (Sigma Chemical Co., St. Louis, Mo.), frozen on dry ice andstored at −70° C. Supernatants were assayed for production of emptyviral particles using an assay for reverse transcriptase which measuresthe incorporation of radiolabeled thymidine into an RNA template. (Goffet al. J.of Virol. 38:239-248 (1981)).

Clones with the strongest reverse transcriptase signals followingautoradiography were thawed, grown up and tested for virus productionfollowing transient transfection. Transfections were done as previouslydescribed, using 10 μg 43.3PGKF3. Media was changed 18 hourspost-transfection. After 24 hours the viral supernatants were collected,filtered through 0.45 μm filters and flash frozen on dry ice. Viralsupernatants were assayed on 3T3 cells plated at 5×10⁵ per 10 cm plate24 hours prior to infection. Infections were done as described above.Cells were then harvested, stained with OKT4A anti-CD4 monoclonalantibody (Ortho Diagnostic Systems Inc., Raritan, N. J.), and analyzedby flow cytometry as described above. Clones displayed varying amountsof packaging function. Those clones with the highest transient titerwere selected for further characterization (Table 5).

TABLE 5 Transduction Efficiency (% CELLS/CLONE # VECTORS CD4 + 3T3Cells) tsa54 none 1.02 tsa54/107.14 ampac + 43.2 2.53 tsa54/107.17ampac + 43.2 0.67 tsa54/107.18 ampac + 43.2 29.76 tsa54/107.22 ampac +43.2 1.76 tsa54/107.24 ampac + 43.2 6.47 tsa54/107.25 ampac + 43.2 0.61tsa54/107.26 ampac + 43.2 1.70 tsa54/107.49 ampac + 43.2 0.89tsa54/107.57 ampac + 43.2 1.12 tsa54/107.73 ampac + 43.2 1.33tsa54/107.75 ampac + 43.2 13.18 tsa54/107.142 ampac + 43.2 0.98293/90.74 ampac + 43.2 24.80 293/90.85 ampac + 43.2 15.06

Clones 90.74, 107.75, and 107.18 were carried for extended time inculture to study ability to maintain the packaging genome over time inthe presence or absence of G418. Cells were split 1:10 to 1:20 every 3to 4 days. At passages 1, 6 and 12 cells were transfected with 10 μg43.2 as described above, and transient viral supernatants were analyzedby infection of 3T3 cells as described above. Of the three clonesstudied, only one (90.74) appeared to have consistent titer over 12passages (Table 6). It also appeared that titer did not depend oncontinued G418 selection. Clone 90.74 has a transient titer equivalentto approximately 107/ml. Clone 90.74 has been deposited with the ATCC,12301 Parklawn Drive, Rockville, Md., under the Budapest Treaty, and hasthere been identified as follows:

Cell Line ATCC Accession No. Deposit Date 90.74 CRL11654 Jun. 10, 1994

TABLE 6 tsa AMPAC STABILITY TEST Transduction Efficiency (% CD4 + 3T3Cells/0.25 ml supernatant) CLONE VECTORS p1 p6 p12 tsa none  0.10 1.20tsa Ampac  0.10 4.06 tsa Ampac + 43.2 63.90 30.97 66.94 107.18 + G418Ampac + 43.2 41.50 22.35 22.68 107.18 Ampac + 43.2 — 23.50 26.22107.75 + G418 Ampac + 43.2 15.42 12.18 4.36 107.75 Ampac + 43.2 — 11.172.05  90.74 + G418 Ampac + 43.2 34.83 30.79 27.79  90.74 Ampac + 43.2 —28.40 31.32

Supernatants were also analyzed for production of RCR by a S+/L-assay onPG4 cells. PG4 cells are Moloney sarcoma virus-transformed cells fromcat brain (ATCC CRL2032). When infected with competent murineretrovirus, PG4 cells produce discernable foci which can bedistinguished microscopically (Haapala, et al. J. of Virol., 53 (3):827-833). PG4 cells were seeded at 5×10⁶ on 10 cm plates 24 hours priorto infection. Infections were done with 1 ml of test supernatant and 4mls of media containing 8 μg/ml polybrene. Media was changed 24 hourslater, and then replaced every 2-3 days until foci developed on positivecontrol plates. All clones studied remained RCR-free through 12passages.

Unexpectedly, these results demonstrate that using the retroviralvectors of the invention, stably transfected 293-derived cell lines thatproduce gag, pol and env proteins were generated. The virus productionfrom these cell lines was equivalent to that produced from transientco-transfection of packaging and retrovirus vectors. Moreover,surprisingly, in the absence of drug selection, these cell linesmaintained production of gag, pol and env proteins. Previous attempts togenerate 293-based retroviral producers using retrovirus constructsdescribed in the literature have failed (Pear et al. Proc. Nat'l. Acad.Sci. (USA) 90:8392-8396 (1993)). After extended passage in culture thesepackaging cell lines do not spontaneously generate replication competentretrovirus.

EXAMPLE VI Production of Double Genome Stable Packaging Cells

This example describes the construction and use of two genomes in 293 ortsA54 packaging cells. First a gag/pol clone was created in human tsa54cells. Cells at 1×10⁶ per 0.8 ml of PBS were co-electroporated with 15μg notI linearized gag/pol ATG (described above) and 1 μg MC1 neo.Electroporation was done at 960 μF and 260 mV (Shigekawa and Dower,(1988) supra) on a Gene Pulser (Biorad, Richmond, Calif.). Cells wereimmediately plated on a 10 cm plate in DME, 1 g/l glucose, 10% donorcalf serum for 48 hours. Cells were then split 1:5, 1:10, 1:20 and 1:50in 1.0 mg/ml G418 selection. Media was changed every 3 days, and after12 days of selection in G418 clones were picked to 24 well plates. Oncecells were confluent, media was collected, filtered through a 0.45 μmfilter and flash frozen on dry ice. Clones were trypsinized and frozenat −70° C. Supernatant was thawed and analyzed for reverse transcriptaseactivity (Goff et al., (1981) supra). Those clones displaying thehighest RT activity were grown up and evaluated for transient virusproduction by calcium phosphate transfection of 5 μgpIK6.1MCVamenvATGUTΔ described above and 10 μg pRT43.2F3 describedabove. Media was changed after 18 hours, and after an additional 24hours the viral supernatants were collected, filtered and frozen foranalysis by infection of 3T3s. Transient virus titers were comparable tothe transient virus titer of the single genome pack line 90.74transfected with pRT43.2F3. and approximately 50% of the viral titerfollowing co-transfection of tsA54 cells with pIK6.1MCVampacUTΔ andpRT43.2F3 (Table 7).

TABLE 7 GAG/POL TRANSIENT TEST Transduction Efficiency (% CLONE VECTORSCD4 + 3T3 Cells) tsa 0.02 293/90.74 ampac + 43.2 28.50 tsa54/111.3gag/pol + 43.2 11.72 tsa54/111.8 gag/pol + 43.2 13.30 tsa54/111.44gag/pol + 43.2 20.29 tsa54/111.89 gag/pol + 43.2 17.93 tsa54/111.4gag/pol + 43.2 32.95 tsa54/111.47 gag/pol + 43.2 19.53 tsa54/111.25gag/pol + 43.2 21.18 tsa54/111.45 gag/pol + 43.2 14.22 tsa54/111.43gag/pol + 43.2 23.75 tsa54/111.22 gag/pol + 43.2 24.27

The four best clones were selected for long term stability studies withand without G418 selection. They were also assayed on PG4 cells for theproduction of RCR and are RCR negative.

Clone 111.4 is co-transfected with pIK6.1MCVamenvATGUTΔ and sv2his(Hartman and Mulligan, Proc. Nat'l. Acad. Sci. (USA) 85:8047-8051(1988)) selected in histinol as described. Clones are picked andcharacterized for virus production by transient transient transfectionas described above. Several high titer clones are characterized forstability and RCR as described.

Packaging lines can be created by replacing the amphotropic env gene inpIK6.1MCVamenvATGUTΔ with other retroviral envelopes, for example,ecotropic, xenotropic, polytropic, MLV, 10A1, Gibbon ape leukemia virus,feline leukemia virus C, Vesicular stomatits virus (VSV) G protein,human T cell Leukemia (HTLV) I and II, and combinations thereof, usingthe methods described above.

EXAMPLE VII Construction of Packaging Plasmids and Production of Virusesof Different Tropisms

a) Xenotropic Packaging Plasmid Constructions

This example describes the construction of packaging plasmids whichencode a xenotropic envelope protein to allow the production ofretroviruses with expanded host range. The envelope protein for thesepackaging plasmids was derived from xenotropic NZB virus (O'Neill etal., J. Virol., 53 (1): 100-106 (1985))

pIK6.1MCVxenopac contains gag/pol from pIK6.1MCVampacUTΔ as well as thexenotropic envelope protein. It was constructed by replacing the 4061Sal1-Nhe fragment of pIK6.1MCVampacUTΔ (described previously in thedetailed description) with the 4200 base pair Sal1-Nhe1 fragment fromthe NZB9-1 xenotropic virus(O'Neill et al., supra).

pIK6.1MCVxenopacUTΔ encodes the ecotropic MMLV gag/pol gene and the NZBxenotropic envelope coding region linked to the SV40 polyadenylationsequence. This plasmid was constructed by deleting untranslatedsequences 3′ from the envelope gene of pIK6.1MCVxenopac by performing aPCR reaction using pIK6.1MCVxenopac as the template with syntheticoligonucleotides 5′ GACCACACTGGCGTAGTAAG 3′(SEQ ID NO 33) and 5′GAATTCGCTAGCTTATTCACGCGATTCTACTTC 3′(SEQ ID NO 34). The resulting 340base pair fragment was digested with BstB1 and Nhe 1 and the 250 basepair product was isolated and used to replace the 312 base pairBstB1-Nhe 1 fragment of pIK6.1MCVxenopac.

pIK6.1MCVxenoenvUTΔ is a packaging plasmid which encodes only the NZBxenotropic envelope as a packaging function. This plasmid wasconstructed as follows: The ATG at the translational start of thexenotropic env gene was converted to an Nco1 site by PCR usingpIK6.1MCVxenopacUTΔ as the template with synthetic oligonucleotides 5′GAATTCCATGGAAGGTTCAGCGTTCTC 3′(SEQ ID NO 35) and 5′ CGTTAGCTGTTTGTCCTGTC3′(SEQ ID NO 36) followed by digestion with Nco1 and Bgl11. Theresulting 120 base pair fragment was purified and ligated in a 4 partligation with a 450 base pair Bgl11-EcoR1 fragment frompIK6.1MCVxenopacUTΔ, a 4541 base pair EcoR1-Hind111 frompIK6.1MCVxenopacUTΔ and a 916 base pair Hind111-Nco1 fragment frompIK6.1MCVgag/pol ATG to produce pIK6.1 MCVxenoenvUTΔ.

b) Ecotropic Packaging Plasmid Constructions

This example describes the construction of packaging plasmids whichencode an ecotropic envelope protein from ecotropic MMLV (Shinnick etal., Nature, 293:543-548 (1981)).

pIK6.1MCVecopac contains the gag/pol genes as well as the ecotropicenvelope protein. It was constructed by replacing the 4175 base pairSal1-Nhe1 fragment of pIK6.1MCVampac with the 4141 basepair Sal1-Nhe1fragment from ecotropic MMLV described previously in the detaileddescription.

pIK6.1MCVecopacUTΔ contains a deletion in the sequences 3′ of theenvelope gene. It was constructed by replacing the 4003 Sal1-Cla1fragment of pIK6.1MCVampacUTΔ with the 3969 base pair Sal1-Cla1 fragmentfrom pIK6.1MCVecopac.

pIK6.1ecoenvUTΔ is a packaging plasmid which encodes only the ecotropicenvelope as a packaging function. It was constructed by replacing the1405 base pair Hha1-Cla1 fragment of pIK6.1amenvATGUTΔ with the 1912base pair Hha1-Cla1 fragment from pIK6.1MCVecopac in a 3-way ligationwith a 3368 base pair Cla1-Spe1 fragment and a 889 base pair Spe1-Hha1from pIk6.1amenvATGUTΔ.

c) Polytropic Packaging Plasmid Construction

This example describes the construction of a packaging plasmid whichencodes a polytropic envelope protein. pIK6.1MCVpolypac contains apol/env fragment from polytropic MCF 247 virus (Holland et al., J.Virol., 47:415-420 (1983)). It was constructed by replacing the 4061Sal1-Nhe 1 fragment of pIK6.1MCVampacUTΔ with the 4200 base pairSal1-Nhe1 fragment from MCF 247 (Holland et al., supra).

d) 10A1 Packaging Plasmid Construction

This example describes the construction of a packaging plasmid whichencodes an amphotropic envelope protein. pIK6.1MCV10A1pac contains apol/env fragment from a recombinant amphotropic MMLV isolate, 10A1 (Ottet al., J. Virol., 64 (2): 757-766 (1990)). It was constructed byreplacing the 4003 Sal1-Cla1 fragment of pIK6.1MCVampacUTΔ with the 4000base pair Sal1-Cla1 fragment from pB6 (Ott et al., supra).

e) Supernatant Transduction of a Wide Variety of Cell Types

This example demonstrates that supernatants from 293 derivativestransiently transfected with pRT43.2F3 and packaging plasmids expressingvarious viral envelopes of a variety of tropisms can efficientlytransduce a variety of other mammalian cells in addition to 3T3 (mousefibroblasts) and human T cells described in the previous examples. Othercells tested include CF2Th (dog thymus), 143B (human osteocarcoma),HT1080 (human fibrosarcoma ), M. dunni (mouse fibroblasts) and 293(human embryonic kidney).

In Table 8 below 1×10⁶ of the indicated cells were transduced with 1 ml(* 10 ul) of supernatant from 293 derivatives transiently transfectedwith pRT43.2F3, and the indicated packaging plasmid.

TABLE 8 3T3 CF2Th 143B HT1080 M. dunni 293 titer Packaging Plasmid (%transduction) (% transduction) (% transduction) (% transduction) (%transduction) (% transduction) pIK6.1MCVampacUTΔ 73 76 76 78 79 58pIK6.1MCVxenopac 2.7 36 50 66 67 65 pIK6.1MCV10Alpac 63 50 71 68 75 60pIK6.1MCVpolypac 40 1.3 4.0 3.1 64 23 pIK6.1MCVecopac* 10 2.2 nd nd 7.2nd nd = not determined

As shown in examples a) throught e), the above described plasmidsencoding envelope genes of a variety of tropisms can be substituted forplasmids encoding an amphtrophic envelope and used in the instantinvention to generate retroviral particles capable of infecting a widevariety of mammalian cell types.

EXAMPLE VIII Production of Stable 293 Viral Producer Clones

This example describes the construction of stable 293 viral producerclones. These stable producer clones can be created from stablepackaging clones either by transfection with retroviral vector or byinfection with retrovirus.

In the first method (transfection with a retroviral vector), the 293amphotropic packaging clone 90.74, described in Example V, was plated at6.5×10⁵ per 10 cm plate in DME (JRH Biosciences, Lenexa, Kans.), 1 g/lglucose, 10% Donor calf serum (JRH Biosciences, Lenexa, Kans.) 48 hoursprior to transfection. 10 μg pRT43.2F3 (a retroviral vector containingthe CD4/zeta chimeric receptor described supra) and 0.5 μg SV2 hyg werecotransfected by calcium phosphate precipitation (Wigler et al. Cell16:777 (1979)). pSV2hyg was derived from pSV2DHFR (Subramani et al. Mol.and Cell. Biol. 1:854-864 (1981)) in the following fashion. First,pSV2DHFR was digested with Hind III and filled in with the Klenowfragment of DNA polymerase I (New England Biolabs, Beverly Mass.) and amixture of the four deoxynucleotide triphosphates. Next, BgII 8-merlinkers (New England Biolabs, Beverly Mass.) were ligated to the bluntends, followed by Bg1II digestion to remove extra linkers and the DHFRcDNA. The vector backbone was isolated and ligated to the Bg1II/Bam HIfragment encoding the hygromycin phospho-transferase gene from pTG4(Giordano and McAllister Gene 88:285-288 (1990)), resulting in pSV2hyg.Eighteen hours post-transfection the media was changed. Twenty fourhours later the cells were split to duplicate plates of 1:10, 1:20, 1:50and 1:100 in media plus 200 μg/ml hygromycin B (Boehringer Mannheim,Indianapolis, Ind.). Media was changed every 3 to 4 days for 14 days.Clones were picked to 24 well plates and grown to confluence. Media wascollected from wells, filtered through a 0.45 μ filter and flash frozenon dry ice. Cells were resuspended in media plus 10% DMSO (SigmaChemical Co., St. Louis, Mo.), frozen on dry ice and stored at −70° C.

The second method for the creation of stable producer clones is byserial infection of the stable packaging clone with a transientsupernatant containing viral particles that display a tropism differentfrom that of the stable packaging clone. Tsa54 cells were plated at6.5×10⁵ per 10 cm plate for 48 hours prior to transfection. 5 μg ofpIK6.1MCVxenopacUTΔ and 10 μg of pRT43.2F3 were cotransfected by calciumphosphate precipitation as previously described. Eighteen hourspost-transfection the media was changed and 24 hours later the media wascollected, filtered through 0.45 μ filters and frozen on dry ice. The90.74 amphotropic packaging clone was plated at 2×10⁶ cell per 10 cmplate and 24 hours later infected with 2 ml of the transientsupernatant, 3 mls of medium and 8 μg/ml of polybrene. Twenty four hourslater the media was changed and the cells were grown to confluence, atwhich time the cells were split 1:10 and grown to confluence. The cellswere subsequently serially re-infected as above with the xenotypicenvelope-containing retrovirus in the transient supernatants, for atotal of 8 serial infections. After eight serial infections thepopulation was cloned by limiting dilution in 96 well plates. Cloneswere transferred to 24 well plates, grown to confluence, and thesupernatants filtered with 0.45 μ filters and frozen on dry ice. Thecells were resuspended in media plus 10% DMSO, frozen on dry ice andstored at −70° C.

Supernatants from clones produced by either method were assayed forviral particles containing CD4/zeta by viral RNA dot blots as follows.Lysis buffer was added to thawed supernatants for final concentrationsof 500 μg/ml. proteinase K (Boehringer Mannheim, Indianapolis, Ind.),100 μg/ml. tRNA (Sigma, St. Louis, Mo.), 2.5 mM EDTA pH 7.5 and 0.5% SDSand incubated for 45 min at 37° C. The lysate was then extracted with anequal volume of phenol, followed by an equal volume of chloroform. Thelysate was split to 2 samples, brought to a final concentration of 375mM NaCl and vortexed 1 ml of chilled ethanol was added to each sampleand the RNA was precipitated over night at −70° C. The lysate was thawedand spun at full speed in a microfuge for 10 minutes. The supernatantwas discarded, the RNA pellet drained, dried, and then resuspended in 20μl 2 mM EDTA pH 7.5, vortexed and heated for 5 minutes at 65° C. 37.5 μlof formamide (USB, Cleveland, Ohio) and 12.5 μl of formaldehyde(Mallinckrodt Chemical, Paris, Ky.) were added and the solution wasincubated at 50° C. for 20 minutes. Finally 100 μl of filtered 10×SSC(1.5 M NaCl, 150 mM sodium citrate pH 7.0) was added and the sampleswere spotted on a nylon filter through the use of a dot blot apparatus.The wells were washed twice with 10×SSC. The blotter was thendismantled, and the RNA was UV crosslinked to the nylon at 1600 μJ(Stratagene, San Diego, Calif.). The filter was hybridized with aDIG-labelled (digoxigenin, Boehringer Mannheim, Indianapolis, Ind.)CD4/zeta probe, and hybridization detected using the Boehringer MannheimGenius System. Clones with the strongest CD4/zeta signals were assayedfor viral titer on 3T3 cells and for RCR as previously described.

Supernatants were assayed for RCR by S+/L-PG4 assay as describedpreviously in Example V. At passage 13 the clones were expanded to a 850cm² roller bottle and supernatant collected at confluence. The entiresupernatant was inoculated on Mus dunni cells, which were grown for 2passages and their final supernatants were then assayed for RCR by PGAS+/L-assay. The clones were all negative for RCR.

Clones with the highest titers were chosen and further characterized forstability of virus production by passaging the clones twice weekly forsix weeks, plus and minus the appropriate selection drug (hygromycin Bfor the transfected clones, G418 for the infected clones). Viral titerson 3T3 cells are shown in Table 9 below for passages 1, 6 and 12 (p1, 6and 12).

TABLE 9 Stability of CD4/Zeta Producer Clones Derived from 90.74 (Viraltiters = % CD4 + 3T3 cells/0.01 ml supernatant/10⁶ cells) method of p1titer × p6 titer × p12 titer × clone producing clones 10⁶/ml 10⁶/ml10⁶/ml 143.15 transfection 1.94 5.05 3.65 143.15 + Hyg transfection ND4.91 4.47 143.43 transfection 0.85 3.38 2.88 143.43 + Hyg transfectionND 3.94 2.80 143.64 transfection 1.37 4.42 3.26 143.64 + Hygtransfection ND 4.27 3.79 143.84 transfection 1.56 3.70 2.65 143.84 +Hyg transfection ND 5.50 3.40 143.86 transfection 2.59 5.50 4.45143.86 + Hyg transfection ND 4.02 2.90 143.90 transfection 1.53 4.002.53 143.90 + Hyg transfection ND 2.73 2.15 method of producing p1 titer× p4 titer × p8 titer × p12 titer × clone clones 10⁶/ml 10⁶/ml 10⁶/ml10⁶/ml 142H.15 infection 24.30 14.78 11.72 13.57 142H15 + infection ND16.56 15.04 14.04 G418 142H.34 infection 9.43 11.17 8.63 8.20 142H.34 +infection ND 9.89 12.10 10.00 G418 142H.62 infection 19.61 16.63 12.4413.23 142H.62 + infection ND 15.16 14.05 11.62 G418 142H.69 infection7.47 10.44 8.69 7.43 142H.69 + infection ND 9.62 11.90 9.48 G418

Table 9 demonstrates that both methods for creating producer clones (viatransfection or infection) resulted in clones that had stable virusproduction for 6 weeks both in the presence or the absence of selection.Also, the 142H and 143 clones were RCR-free (data not shown). The 3T3titers of the infection amplified clones (142H) were 2-3 fold greaterthan the transfected clones (143).

The producer clone supernatants were further characterized bytransducing human primary CD8+ T cells that were isolated as previouslydescribed. CD8+ cells were plated at 10⁶ cell/ml/well of a 24-well platein AIM V+100 Cetus units/ml IL-2 (Chiron, Emeryville, Calif.) 24 hoursprior to transduction. For transduction the cell volume was reduced to0.5 ml and 0.75 ml of appropriate supernatant and 0.75 ml of AIM V+200Cetus units/ml IL-2+4 μg/ml polybrene were added for 4-24 hours at 37°C. This was done once a day for three days. After the third day cellswere returned to growth media (50% RPMI, 50% AIM V, 5% human serum(Sigma Chemical Co., St. Louis, Mo.)) for an additional three days andthen analyzed for surface CD4 as described previously. The 293supernatants were compared with supernatant from 78.81, a CD4/zetaproducer clone generated by amplification cocultivation (Bestwick etal., Proc. Natl. Acad. Sci., USA, 85:5404-5408 (1988)) from the3T3-based PA317 packaging line (Miller et al, U.S. Pat. No. 4,861,719).Table 10 below demonstrates transduction efficiency of CD4/zeta producerclones on human CD8+ T lymphocytes.

TABLE 10 Human Primary CD8 + T Cell Transduction by Supernatants ofTransfected or Infected Amplified Clones T cell trans- packaging ductionClones clone method 3T3 titer × 10⁶/ml (% CD4+) 78.81 PA317 Infection2.86 13.97 142H.15 90.74 Infection 13.16 60.55 142H.34 90.74 Infection6.14 45.44 142H.62 90.74 Infection 11.32 88.63 142.69 90.74 Infection5.22 48.99 143.15 90.74 Transfection 3.5 42.65 143.43 90.74 Transfection1.87 14.26 143.64 90.74 Transfection 3.5 51.48 143.84 90.74 Transfection2.11 22.66 143.86 90.74 Transfection 3.66 58.14 143.90 90.74Transfection 3.69 41.87

The 293-based clones consistently provides higher titer on the 3T3 cellsand a higher level of transduction of the T cells than the 3T3 basedproducer, 78.81. These transduction results are 5 to 10-fold greaterthan those previously reported in the literature for the cocultivationof T cells with producer clones, and 40 to 80-fold greater than the 1-2%T cell transduction by supernatants previously reported (Fauser, J.Cell. Biochem., 45:353-358 (1991), Hwu et al., J. Immunol.,150:4104-4115 (1993)), Imbert et al., Cancer Gene Therapy, 1:259-265(1994)), and Mavillo et al., Blood, 83:1988-1997 (1994)).

Table 10 also demonstrates that the 3T3 titer does not predict T celltransduction. For example, although infection-amplified clone 142H.15has 3-fold greater 3T3 titer than transfected clone 143.64, they haveequivalent T cell transductions of 60.55% and 58.14%, respectively.

EXAMPLE IX Construction of Stable Two Genome 293 Packaging Line

This example describes the creation of stable clones of 293 cells thatcontain two helper sequences encoding packaging functions. Applicantsfirst constructed a stable gag/pol clone in 293 cells that was then usedfor the production of a variety of packaging clones with the differentenvelope encoding plasmids of Example VII.

To construct the gag/pol clone, 293 cells were plated at 6.5×10⁵ for 48hours, then cotransfected with 10 μg pIK6.1MCVgag/polATG and 1 μg MC1neo(Stratagene, La Jolla, Calif.) by calcium phosphate precipitation aspreviously described. Medium was changed after 18-24 hours, and after anadditional 24 hours the cells were diluted 1:10, 1:20, 1:50, 1:100,1:500 and 1:1000 into 1mg/ml G418 selection. Cells were fed every 3-4days, and after 12 days clones were picked to 24-well plates. Once thecells were confluent, the medium was collected, 0.45 μ filtered andfrozen on dry ice. The cells were frozen in medium plus 10% DMSO andstored at −70° C. Supernatant was thawed and analyzed for reversetranscriptase activity (Goff et al, supra). Those clones displaying thehighest reverse transcriptase activity were grown and evaluated fortransient virus production by transfecting with 5 μg ofpIK6.1amenvATGUTΔ and pRT43.2F3 as described above. Supernatants werecollected and assayed for CD4 titer by infection of 3T3 cells aspreviously described. The four clones with the best transient titerswere studied for long term stability. Clones were passaged twice a weekfor 6 weeks, with and without G418 selection. Transient transfectionswere done at passage (p) 1, 5, 9 and 13 with pIk6.1amenvATGUTΔ andpRT43.2F3, and the supernatants were evaluated for titer on 3T3 cells.(Table 11).

TABLE 11 Stability of packaging cell clones encoding gag/pol p1 titer ×p5 titer × p9 titer × p13 titer × clone 10⁶ 10⁶ 10⁶ 10⁶ 35.32 0.73 2.011.29 1.53 35.32 + G418 ND 1.95 1.77 1.60 35.35 0.21 0.87 1.23 1.1135.35 + G418 ND 0.74 1.14 0.92 35.74 0.46 0.99 1.11 0.64 35.74 + G418 ND1.07 1.90 1.07 35.88 0.30 0.64 0.47 0.28 35.88 + G418 ND 0.59 0.34 0.15

Virus production from the clones was stable over 13 passages in both thepresence and absence of G418. Clone supernatants were also evaluated forRCR by S+/L-PG4 assay as previously described in Example V and found tobe negative.

Gag/pol clone 35.32 was selected for further transfection with envelopeplasmids since it was determined to be stable and RCR-free in long termpassage, and had the best growth characteristics. Clone 35.32 was platedat 6.5×10⁵ 48 hours prior to transfection. Ten μg ofpIK6.1amenvATGUTΔ+0.5 μg of SV2 hyg were cotransfected, media waschanged at 18-24 hours, and after an additional 24 hours cells weresplit into 200 μg/ml hygromycin B (Boehringer Mannheim, Indianapolis,Ind.). Medium was changed every 3-4 days and at day 14 clones weretransferred to 24 well plates. Once the cells were confluent, the mediumwas collected, 0.45 μ filtered and frozen on dry ice. Cells from eachclone were divided in half, and half were frozen down in medium +10%DMSO and stored at −70° C. The other half of the cells were thenanalyzed for envelope protein production using rat anti-gp70 antibody83A25 (Evans et al., J. Virol., 64:6176-6183 (1990)). Cells weretrypsinized, washed with PBS plus 2% FBS, incubated with 47 μg antibodyat 4° for 30 min, washed three times with PBS/FBS, incubated with goatanti-rat IgG-PE at 0.5 μg/tube (Biosource International, Camarillo,Calif.), washed two times and resuspended in 0.1% formaldehyde. Cellswere then analyzed by flow cytometry. Supernatants were thawed andanalyzed for reverse transcriptase activity (Goff et al, supra). Clonespositive for gp70 and with the highest reverse transcriptase levels weregrown up and transiently transfected with 10 μg of pRT43.2F3 asdescribed, and assayed for CD4 titer on 3T3 cells. The clones with thehighest titers were passaged twice a week for six weeks with and withouthygromycin B. Transient transfections of 10 μg of pRT43.2F3 were done atp1 and p5 (Table 12). Clone supernatants were also negative for RCR byS+/L-PG4 assay.

TABLE 12 Stability of Two Genome Packaging Clones gag/pol + amenv clonesp1 titer × 10⁶ p5 titer × 10⁶ 37S2.8 1.01 1.39 37S2.18 0.35 0.60 37P2.40.30 0.90 37P2.9 0.19 0.34

Table 12 demonstrates that packaging clones containing the gag/pol andamphotropic envelope genes can be isolated which stably produce hightiter retroviral supernatants. Two genome packaging clones containing axenotropic envelope gene were also constructed as described above. Inthis case, 10 μg of pIK6.1CMVxenoenvUTΔ+0.1 μg pIKpur was cotransfected.PIKpur was constructed by the insertion of a 600 base pair cDNA encodingStreptomyces alboniger puromycin-N-acetytransferase (abbreviated pa,GenBank Accession No. M25346 nucleotides 254-853) into pIK6.1, describedpreviously. Cells were selected in 0.5 μg/ml puromycin (Sigma, St.Louis, Mo.). Clones were then isolated, assayed for reversetranscriptase activity and envelope protein as above, and the clonespositive for gp70 with the highest reverse transcriptase levels arechosen for long term stability studies. These clones are also evaluatedfor virus production by transient transfection of 10 μg of pRT43.2F3.Those clones with the highest transient titers are maintained for sixweeks in culture with and without puromycin, and assayed for virusproduction at passages 1, 5, 9 and 13 after transient infection withpRT43.2F3, as described above, to determine the stability of packagingfunction.

EXAMPLE X Construction of a Retroviral Vector and Packaging PlasmidsContaining the RSV Enhancer and Promoter

This example describes the construction of a retroviral vector whereinthe 5′ LTR of the retroviral vector contains the enhancer and promoterfrom the U3 region of the Rous Sarcoma Virus (RSV) joined to the Rregion of MMLV and the construction of packaging plasmids wherein thepackaging functions are encoded by two plasmid based expression vectorsin which expression is under the control of the enhancer and promoterfrom the U3 region of the Rous Sarcoma Virus (RSV).

pRT43.RSV.F3 is a retroviral construct in which the enhancer andpromoter of RSV is joined to the R region of MMLV as follows: pIK 6.1RSVwas derived from pIK6.1 by replacing the 679 base pair HindIII-XbaI CMVIE enh/pro fragment of pIK6.1 with a 235 base pair HindIII-Xba I RSVenhancer/promoter fragment generated by PCR using pREP4 (InvitrogenCorp. San Diego, Calif.) as a template with synthetic oligonucloetides5′-GAATTCAAGCTTAATGTAGTCTTATGCAAT 3′(SEQ ID NO. 37) and 5′GAATTCTCTAGAGTTTATTGTATCGAGCTA 3′ (SEQ ID NO. 38), followed by digestionwith HindIII and XbaI. pRT43.RSV.F3 was derived from pRT43.2F3 byreplacing the 710 base pair HindIII-Asp718 fragment of pRT43.2F3 with a219 base pair HindII-TaqI fragment from pIK6.1 RSV and a 46 base pairTaqI-Asp718 synthetic oligonucleotide (consisting of oligonucleotides 5′CGATACAATAAACGCGCCAGTCCTCCGATTGACTGAGTCCCCGG 3′ (SEQ ID NO. 39) and 5′GTACCCGGGCGACTCAGTCAATCGGAGGACTGGCGCGTTTATTGTAT 3′(SEQ ID NO. 40) in afour-part ligation with a 1006 base pair Asp718-Bgl II fragment frompRT43.2F3 and a 6897 base pair Bgl II-HindIII fragment from pRT43.2F3.

pIK6.1RSVgag/polATG is a packaging plasmid encoding the gag/pol genesunder the control of the RSV U3 region. It was derived frompIK6.1gagpolATG by replacing the 1258 base pair AflIII-NsiI fragment ofpIK6.1gagpolATG with the corresponding 806 base pair fragment from pIK6.1RSV in a two part ligation.

pIK6.1RSVamenvATGUTΔ is a packaging plasmid encoding the amphotropicenvelope gene under the control of the RSV U3 region. It was derivedfrom pIK6.1amenvATGUTΔ by replacing the 1373 base pair AflIII-Bgl IIfragment of pIK6.1amenvATGUTΔ with the 1085 base pair AflIII-BglIIfragment of pIK6.1RSV in a three-part ligation with the 2714 base pairBglII-DraIII fragment and the 2033 base pair DraIII-AflIII fragment frompIK6.1amenvATGUTΔ.

The following table (Table 13) compares the titers of retroviralparticles when the enhancer/promoter regions of the retroviral vectorsand packaging plasmids are derived from RSV, MMSV or CMV. These resultsdemonstrate that the vectors and packaging plasmids containing RSV LTRsare as efficient as those containing MMSV or CMV LTRs.

TABLE 13 Comparison of Retroviral Production in tsA54 Cells using RSV,MMSV or CMV Enhancer/Promoter Vectors Enhancer-Promoter/ Titer/mlsupernatant Retroviral Vector Packaging Protein on 3T3 cells (expt. 1)pRT4.3.2F3(CMV) MCV/gagpol 3.4 × 10⁶ MCV/amenv pIKT4.2F3 (MMSV)MCV/gagpol 5.3 × 10⁶ MCV/amenv pRT43.RSVF3 MCV/gagpol 6.9 × 10⁶MCV/amenv (expt. 2) pRT42.2F3 (CMV) MCV/ampac 6.1 × 10⁶ pIKT4.2 F3(MMSV) MCV/ampac 1.3 × 10⁷ pRT43.RSVF3 MCV/ampac 1.5 × 10⁷ (expt. 3)pRT43.2F3 (CMV) MCV/gagpol 4.6 × 10⁶ MCV/amenv pRT43.2F3 (CMV)MCV/gagpol 4.8 × 10⁶ MMSV amenv pRT43.2F3 (CMV) MCV/gagpol 3.2 × 10⁶RSV/amenv pRT43.2F3 (CMV) RSV/gagpol 1.6 × 10⁶ MCV/amenv pRT43.2F3 (CMV)RSV/gagpol 1.7 × 10⁶ MMSV/amenv pRT43.2F3 (CMV) RSV/gagpol 2.3 × 10⁶RSV/amenv pIKT4.2 F3 (MMSV) RSV/gagpol 2.4 × 10⁶ RSV/amenv (expt. 4)pRT43.2F3 (CMV) MCV/gagpol 6.5 × 10⁶ MCV/amenv pRT43.RSVF3 RSV/gagpol5.1 × 10⁶ RSV/amenv

EXAMPLE XI High Level Supernatant Transduction of Human CD8+ T Cells

In this example, Applicants demonstrate the high level transduction ofCD8+ T-cells from different donors with retroviral supernatants from293-derived cells which were either stably or transiently transfectedwith the retroviral vectors and packaging plasmids of the instantinvention. Transduction efficiencies of retroviral supernatants fromtransient and stable 293-derived cells are compared with supernatantsfrom 3T3-derived stable packaging cells.

For transient viral production, tsA54 cells are seeded at 0.6×10⁶/10 cmplate 48 hrs prior to CaPO₄ transfection with 5 ug of packaging and 10ug of retroviral plasmid. Twenty four hours post transfection, the mediais exchanged for fresh media. Forty eight hours posttransfection,supernatant was harvested, filtered through 0.45 micronfilters, stored at −70° C. and thawed immediately before use.

The supernatants were characterized by transducing human primary CD8+ Tcells that were isolated as previously described. CD8+ cells were platedat 10⁶ cell/ml/well of a 24-well plate in AIM V+100 Cetus units/ml IL-2(Chiron, Emeryville, Calif.) 24 hours prior to transduction. Fortransduction, the cell volume was reduced to 0.5 ml and 0.75 ml ofappropriate supernatant and 0.75 ml of AIM V+200 Cetus units/ml IL-2+4μg/ml polybrene were added for 4-24 hrs at 38° C. After the transductioncells were returned to growth media for an additional 3-20 days andanalyzed for CD4/zeta surface expression as described previously. Table14 summarizes the results when three independent isolates of CD8 T-cellswere transduced with the indicated viral supernatants from transientlytransfected tsA54 cells, stably transfected 293 cells (142H.62) orstably transfected 3T3-derived PA317 cells (78.81).

TABLE 14 High level supernatant transduction of Human CD8+ T cells CD8CD8 CD8 % transducton % transduction % transduction Method retroviralvector packaging vector (Donor 1) (Donor 2) (Donor 3) tsA54 Transientmock mock .1 1.8 1.6 tsA54 Transient pRT43.2F3 pIK6.1MCVampacUTΔ 51 4446 tsA54 Transient pRT43.3PGKF3 pIK6.1MCVampacUTΔ 63 53 50 293Stable142H.62 pRT43.2F3 pIK6.1MCVampacUTΔ 57 53 57 PA317 stable 78.81pRTD4.2svgF3e- 7 9.7 8.8

Table 14 demonstrates that virus-containing supernatants from 293 cells,either stably or transiently transfected according to the methods of theinstant invention transduce CD8+T cells at significant higher frequencythan supernatants from 3T3 cells.

EXAMPLE XII High-Level Transduction of Primary Human Cells

This example describes the use of the constructs of the instantinvention to efficiently transduce primary human CD34+ bone marrow cellsusing a protocol involving supernatant infection. To increase theefficiency of viral infection, purified CD34+ cells were placed ontoplates which were coated with monoclonal antibodies against the adhesionmolecules VLA-4, VLA-5, CD29, CD11a, CD11b, and CD44 prior to infection.This supernatant protocol results in levels of infection which areequivalent to those found with the cocultivation of virus-producing 293cells. The example also describes the use of this method for the highlevel transduction of other primary human cells.

The ability to maintain both self-renewing and differentiatingpopulations of cells derived from stem cells depends upon cell-cellcontact of stem cells and stromal cells in the bone marrow (Gordon andGreaves, Bone Marrow Transplantation, 4:335-338 (1989)). The contact ofstromal cells and hematopoietic stem cells involves many moleculesincluding growth factors, exemplified by the kit ligand on stromal cellsand c-kit receptor found on stem cells (Zsebo et al., Cell, 63:213-224(1990)) and adhesion molecules, fibronectin on stromal cells and VLA-4on hematopoietic stem cells (Williams et al., Nature 352:438-441(1991)). These contact molecules are either transmembrane or, if locatedextracellularly, they are proteins which contact transmembrane proteinsand enable signals for either self-renewal or differentiation to betransmitted between the stromal cells and the stem cells.

In order to improve the poor retroviral gene transfer into hematopoieticstem cells by supernatant infection, recreation of the cell-cellcontacts was attempted and resulted in higher efficiency of genetransfer (Morre et al., Blood 79:1393 (1992)). However, cocultivation ofbone marrow cells on stroma is neither acceptable by the FDA nor is iteconomically feasible. Therefore, attempts at recreation of the cellcell contacts have been undertaken. The interaction of fibronectin onstromal cells and VLA-4 on hematopoietic stem cells (Williams et al.,supra) has been previously demonstrated. By isolating the CS-1 domain offibronectin responsible for this interaction and coating plates withthis protein molecule, Moritz et al. demonstrated that retroviral genetransfer by supernatant infection can be significantly enhanced (J.Clin. Invest., 93:1451-1457 (1994)). This approach necessitates theisolation of significant quantities of proteolytic fragments fromnatural material. Furthermore, many molecules participate in the cellcell interactions of stroma and stem cells (Liesveld et al. Blood81:112-121 (1993)).

We have taken the generalizable approach taken of coating cell cultureplates with antibodies to adhesion molecules that participate instromal-hematopoietic stem cell cell-cell contact either singly or incombination, followed by retroviral gene transfer. We have confirmed theobservations of Moritz et al., (supra) that the fibronectin/VLA-4interaction can enhance retroviral transduction and that purified CS-1fragment can be replaced by anti-VLA4 antibody. We have gone on to showthat not only is the recreation of CS1-VLA4 cell-cell contact effectiveat enhancing retroviral gene transfer but that other cell-cell contactsbetween stromal cells and hemopoietic cells can be recreated usingantibodies to VLA5, CD29, CD11a, CD11b (Liesveld et al. Blood81:112-121, (1993)) and can also improve retroviral transduction.

Cell-cell contact plays an important role for the activation and growthof many cells of the hemapoietic lineage. For example, many cell-cellcontacts have been identified that are essential for T cell activation(Bolhuis et al., Cancer Immunol. Immunother, 34:1-8 (1991)) includingthe interactions of receptor/coreceptor pairs on T lymphocytes andantigen presenting cells such as LFA-1 and ICAM-1, and CD-2 and LFA-3.In B lymphocytes, the CD40/gp39 interaction takes place between Blymphocytes and T lymphocytes and is necessary for B lymphocyteactivation (Armitage et al., Sem. Immunol., 6:267-278 (1994)).Antibodies to CD2 (Springer et al., Nature 323:262 (1987)) or CD40 cansubstitute for the ligands and mediate cell-cell interaction andactivation. The transduction of T and B lymphocytes by supernatantinfection has been reported to be of low efficiency (Hwu et al., J.Immunol., 9:4104-4115 (1993); Baker et al., Nucleic Acids Res., 20:5234(1992)). Using an approach similar to that for stem cells, antibodies tothe receptor present on the target cells (i.e. anti-CD2 or LFA1 antibodyfor T lymphocytes and anti-CD40 antibody for B lymphocytes), which havebeen shown to activate these respective cell types, can also be used toenhance the supernatant transduction efficiency of these cells.

High Level Transduction of Primary Human Hematopoietic Stem Cells

CD34+ cells were isolated from the peripheral blood of patientsundergoing cyclophosphamide and G-CSF treatment. Mononuclear cells areisolated from leucopheresed blood by fractionation using a standardFicoll gradient (Pharmacia, Piscataway, N.J.). The CD34+ cells areisolated using positive selection on a CellPro CEPRATE LC affinitycolumn (CellPro, Bothell, Wash.). Post purification analysis via flowcytometry demonstrates that this population is approximately 90% CD34+.This population of cells is then cultured for a period of 48-72 hours ata density of 0.5-1×10⁶ cells/ml in “prestimulation medium” whichcontains Myeloid Long Term Culture Medium supplied as a complete mediumfrom Terry Fox Labs, (Vancouver, Canada) with the addition of 100 ng/mlhuman Stem Cell Factor (SCF), 50 ng/ml human IL-3, and 10 ng/ml humanIL-6 (Genzyme, Cambridge, Mass.).

Viral supernatant for infection of the CD34+ cells was produced asfollows. 293 cells were transfected by first plating at a density of1.4×10⁶ cells/10 cm dish 24 hours prior to transfection, followed byco-transfection with 10 ug pRT43.2F3 vector DNA (encoding CD4/zeta) and7.5 ug of the packaging plasmid pIK6.1MCVampacUTΔ. Eighteen hours later,transfection media is removed and replaced with 10 mls IMDM (JRHBiosciences, Woodland Calif.)+10% FBS. Viral supernatant is thencollected 24-36 hours later and 100 ng/ml human SCF, 50 ng/ml humanIL-3, 10 ng/ml human IL-6, and 8 ug/ml polybrene were added.

To produce antibody-coated plates, 10 ug of antibody or a combination ofantibodies (Immunotech, Westbrook Me.) is dissolved in 1 ml of PBS andincubated overnight in the tissue culture plates as discussed above.After incubation the plates are washed gently with PBS, and cells andviral supernatant are added immediately. As a comparison, tissue cultureplates were also coated with fibronectin or a chymotryptic fragment offibronectin, CS-1, as reported by Williams et al. (Nature 352: 438-441(1991)) and Moritz et al. (J. Clin. Invest 93: 1451-1457 (1994)).Fibronectin and CS-1 coated plates are made by adding 30 ug/ml PBS offibronectin, derived from human plasma, or CS-1 (Sigma, St Louis, Mo.)to tissue culture plates. The plates are then incubated at 37° overnightand washed with PBS (“24 hour method”). Alternatively, the plate isplaced under UV light for 1 hour with the lid off and then an additionalhour with the lid on, the PBS is removed, one ml of 2% BSA is added for20 minutes, and the plates are washed with DPBS/0.2% HEPES (“2 hourmethod”) (Williams et al. supra).

As shown below in Table 15 Expt. 1, the use of antibody-coated platesdramatically increased the percentage of hematopoietic stem cells whichwere transduced by the retroviral supernatants (3.5% without coatingcompared to from 12.2 to 16.9% with coating with a single antibody). Theuse of a combination of antibodies increased the transduction frequencyeven further (43.2% with anti-VLA-4 and anti-CD44, Expt. 2). Expt. 3demonstrates that the level of transduction with two antibodies iscomparable to that achieved with fibronectin or CS-1 coating (Moritz etal., supra). Applicants have also determined that the use of the “24hourmethod” of fibronectin coating results in consistently greatertransduction frequencies then the “2hour method”)

TABLE 15 Supernatant transduction of CD4/zeta into CD34 + stem cellsCoating of plates % CD4 + cells Expt. 1 None 3.5 anti-VLA-4 16.2anti-VLA-5 12.2 anti-CD29 16.6 anti-CD11a + anti-CD11b 15.1 Fibronectin(24 hours) 16.9 Expt. 2 Fibronectin (24 hours) 31.1 anti-VLA-4 andanti-CD44 43.2 Expt. 3 Fibronectin (2 hours) 29.6 Fibronectin (24 hours)53.8 CS-1 (2 hours) 69.5

Table 15 above also demonstrates that the use of fibronectin plates, incombination with the viruses of the instant invention, results in ahigher efficiency of transduction of stem cells then that previouslyreported for other retroviral systems (Moritz et al. supra).

Supernatants from the stable CD4/zeta virus producer cells describedabove in Example VIII are also efficient transducers of hematopoieticstem cells. In this example, the CD34+ cells are harvested afterpre-stimulation, washed, and plated at a density of 7.5×10⁵ cells/wellin a 6-well tissue culture dish coated with CS-1 as described above (10ug CS-1). For undiluted supernatants, cells are resuspended in 2 mls ofviral supernatant with the addition of cytokines and polybrene, asdescribed above. Viral supernatants were then diluted 1:2, 1:10 and 1:50in medium containing cytokines and polybrene. Four hours afterinfection, the cells were collected, washed, and resuspended in viralsupernatant for additional exposure to the virus overnight. Fresh“pre-stimulation” media was then added to the cells after washing themfree of viral supernatant. The cells were then cultured and analyzed viaflow cytometry for CD4 expression.

As shown in Table 16 below, viral supernatants from stable producers canalso be used to efficiently transduce CD34+ cells. Applicants have alsofound that the level of transduction of CD34+ cells by the viralsupernatants from the various producer clones is correlated with theirability to transduce T cells (Table 10), and not their viral titer asdetermined by infection of 3T3 cells.

TABLE 16 Transduction of hematopoietic stem cells using supertants fromstable producers % transduction of CD34 + Viral titer Viral SupernatantDilution cells on 3T3 cells 293 142H.15 1:1 38.1 1.0 × 10⁷ 1:2 35.6 1:10 24.8  1:50 6.8 293 142H.62 1:1 62.7 1.1 × 10⁷ 1:2 54.9  1:10 29.2 1:50 8.2 293 142.69 1:1 30.4 1.3 × 10⁷ 1:2 26.6  1:10 18.1  1:50 4.8

Viral supernatants from stable producer clones can also be used totransduce CD34+ cells incubated on antibody-coated plates. As describedabove, plates are coated with antibodies to anti-adhesion molecules andthe CD34+ cells are purified and added to the plates. Viral supernatantsare then added and the percentage of transduced cells is determined.

EXAMPLE XIII Episomal Replication of Retroviral Plasmids

In another embodiment of the invention, we obtain high level transientretroviral production using plasmids containing the Epstein-Barr Virus(EBV) EBNA1 and oriP gene sequences. These sequences have been shown todirect multi-copy episomal replication of plasmid sequences for manycell generations (Yates et al., Nature 313:812-815 (1985); Margolskee etal., Mol. Cell. Biol. 8:2837-2847 (1988)). Plasmids containing the EBNA1and oriP sequences along with the retroviral genome may allow formaintenance of multiple copies of these retrovirus-containing plasmidsin the absence of plasmid integration. This invention will alleviate theneed for multiple cross infections of retroviral producer cells withpseudotyped retroviral particles to obtain high titer stable producercell lines (Bestwick et al., supra). The use of this plasmid will alsoeliminate the time required to screen multiple clones to isolate hightiter producer clones. These sequences have the additional benefit ofenabling the generation of high titer producer cell populationscontaining vectors that have internal promoters and deletions in theenhancer or enhancer/promoter regions of the 3′ LTRs, and therefore cannot be amplified by amplification cocultivation or serial infection. Dueto the enhancer deletion in the 3′ LTR in internal promoter vectors,such constructs without the EBV sequence would need to be transfectedinto packaging cell lines followed by screening 50-100 clones in orderto produce high titer retrovirus producer clones. Insertion into theEBV/oriP replicating vectors eliminates the need to screen large numbersof clones and enables rapid isolation of producer populations forinternal promoter vectors.

Vectors containing internal promoters are of particular interest for thefollowing reasons. Upon transduction of some primary cells withretroviral vectors in which the transcription of the gene of interest isdriven from the viral long terminal repeat (LTR), gene expression iseliminated over time in vivo due to methylation of the viral LTR. Oneexample of this behavior has been observed following transduction ofhematopoietic stem cells (Challita and Kohn Proc. Natl. Acad. Sci., USA91:2567-2571 (1994)). In order to overcome this problem, transcriptionalcontrol elements (enhancers, promoters, dominant control elements) canbe introduced internal to the vector. These internal promoters, whichare resistant to inactivation (Lim et al., Mol. Cell. Biol. 7:3459-3465(1987); Wilson et al. Proc. Natl. Acad. Sci, USA 87:439-443 (1990);Correll et al., Blood 84:1812-1822 (1994)), include cellular promoters(human or mouse phosphoglycerate kinase, chicken beta actin) as well asviral promoters (SV40 early region, herpes simplex virus thymidinekinase). These vectors can be constructed with either an intact 3′ LTR(Correll et al., Blood 84:1812-1822 (1994)) or with a 3′ LTR containingan enhancer deletion (for example, Wilson et al. Proc. Natl. Acad. Sci,USA 87:439-443 (1990). The internal promoters enable expression in allof the differentiated cell types derived from a pluripotenthematopoietic stem cell. Other internal promoters can be used toregulate expression specific for a single cell type. For example, thehuman beta globin promoter directs specific expression in murineerythrocytes following stem cell gene transfer (Dzierzak et al., Nature331:35-41 (1988)) and the creatine kinase promoter is specific forexpression in myoblasts (Dai et al., Proc. Natl. Acad. Sci. USA89:10892-10895 (1992)).

This example describes the construction of retroviral plasmidscontaining EBNA1 and oriP sequences. pRT43.3PGKF3CEP4ro is a retroviralvector plasmid containing all necessary elements for high levelproduction of full length packageable retroviral transcripts (a 5′ LTR,a psi site, an internal PGK promoter, a 3′ LTR with an enhancer deletionand the 3′ flanking regions including SV40 poly A site and origin ofreplication (ori)) on a plasmid backbone containing EBV EBNA1 and oriPsequences. The use of the SV40 origin also enables virus titer to betransiently increased by transfection of plasmids encoding SV40 Tantigen, which induces replication via the SV40 origin and increasesplasmid and gene expression (Heinzel et al. J. Virol., 62 (10):3738-3746 (1988)). This vector plasmid was generated in the followingmanner:

pUC.CEP4 was created to enhance bacterial plasmid production byreplacing the 3086 base pair Sal1 fragment of pCEP4 (Invitrogen city,state) with a 2691 base pair Sal1 fragment comprised of a 1371 basepairSal 1-filled Afl111 fragment from pUC19 (New England Biolabs,Beverly, Mass.) and a 1316 base pair blunted Bsm1-Sca1 from pHEBO(Sugden et al. Mol. and Cell Biol. 5:410-413 (1985). (pRT43.3PGK3contains a deletion of sequences in the 3′ LTR which results in the lossof enhancer function. One skilled in the art can produce other 3′ LTRsequences lacking enhancer function for use in the instant inventionusing conventional techniques).

The pRT43.3PGKF3CEP4do vector was created by inserting a 5327 base pairSnaB1-Avr11 fragment from pRT43.3PGKF3 (described above in the detaileddescription) into a 9695 base pair SnaB1-Nhe1 fragment from pUC.CEP4.The pRT43.3PGKF3CEP4ro vector was generated by inserting an 6313basepair Sal 1-Sal1(partial) fragment from pRT43.3PGKF3CEP4do into an8672 base pair Sal1-Sal1 backbone fragment from pUC.CEP4. pRT43.3PGK3contains a deletion of sequences in the 3′LTR which results in the lossof enhancer function, but still allows virus polyadenylation andtransmission. One skilled in the art can produce other 3′LTR sequencelacking enhancer function for use in the instant invention usingconventional techniques.

Retroviral supernatants were produced by transient transfection of thesepUC.CEP4 based plasmids into tsA54 cells along with the pMCVampacUTΔpackaging plasmid described previously. The titer of these supernatantswas determined by infection of 3T3 cells as shown in Table 19.

TABLE 19 Retroviral vector 3T3 titer (expt. 1) 3T3 titer (expt. 2)43.3PGKF3 2.4 × 10⁶ ND 43.3PGKF3CEP4do 2.1 × 10⁶ 6 × 10⁶ 43.3PGKF3CEP4roND 6 × 10⁶

Table 19 shows that the plasmids containing the EBNA1 and oriP sequencesalong with the retroviral genome (pRT43.3PGKF3CEP4do andpRT43.3PGKF3CEP4ro) produce high titer supernatants comparable to thoseproduced with a vector without the EBV sequences (pRT43.3PGKF3).

The EBNA1 and oriP containing vector plasmids can also be packaged inlong-term, stable cell lines, as shown below. To produce these celllines, the 90.74 amphotropic packaging cells were plated 48 hours priorto transfection and 10 ug of 43.3PGKF3CEP4ro was transfected aspreviously described and cells resistant to 200 ug/ml hygromycin B wereselected. Virus-containing supernants from independent bulk populationsof hygromycin-resistant cells were collected and the viral titers weredetermined by infecting 3T3 cells. Viral supernatants were also used toinfect CD8+ T cells and the percent of vector-containing cells wasdetermined by analysing the production of the CD4 antigen encoded by the43.3PGKF3CEP4ro vector. These results are shown below in Table 20.

TABLE 20 TR157 CEP4 Population Titers Bulk Population Number 3T3 Titerx10⁶ 2-10A 0.92 2-10B 0.76 2-20A 1.55 2-20B 1.47 2-50A 0.63 2-50B 1.162-100A 0.68 2-100B 1.21 3-10A 1.1 3-10B 1.08 3-20A 0.65 3-20B 1.52

The above table demonstrates that the EBNA1 and oriP containing plasmidscan be used to rapidly produce virus which can efficiently infect 3T3cells and T cells without the need to isolate stable packaging clones.

Virus-producing clones are produced from the bulk population ofhygromycin-resistant cells by standard procedures. The clones arescreened for production of viral RNA by hybridization analysis using dotblots, and the clones with highest production are selected for furthergrowth and analysis. After a further 6 weeks of selection in hygromycin,viral supernatants are analyzed for the infection of 3T3 cells and Tcells.

EXAMPLE XIV GALV-based Vectors and Packaging Plasmids

Many human cells are not efficiently infected using retroviral vectorsand packaging systems based on MMLV. To aid in circumventing thisproblem, vectors and packaging plasmids can be prepared which are basedon other retroviruses, for example the primate retrovirus GALV. ManyMMLV sequences (LTRs, psi packaging sites, splice/donor and acceptorsites, and/or primer binding sites) may be substituted usingconventional methods in the instant retroviral vectors by the analogousregions from GALV viruses to produce viruses capable of infecting a widevariety of mammalian cells, in particular human cells, when used in thepresent invention. GALV gag and pol genes can also be used in retroviralpackaging plasmids. The production of pseudotyped virions having GALVenvelope proteins has been demonstrated. (Wilson et al., J.Virol.63:2374-2378 (1989)). In addition, Miller et. al., (J. Virol.65:2220-2224 (1991)), describe construction of hybrid packaging celllines that express GALV envelope and MMLV gag-pol proteins.

The construction of retroviral packaging plasmids which contain genesencoding GALV gag/pol or envelope proteins is described below.

pIK6.1GALVSEenv contains the gene encoding the GALV envelope protein. Itwas constructed by replacing the 1980 base pair Bgl11-Nhe1 amphotropicenvelope region of pIk6.1amenvATGUTΔ with the corresponding GALVenvelope encoding region from GALV Seato strain (Kawakami, et al.,Transplant Proc., 6:193-198 (1974)). A PCR reaction was performed withsynthetic oligonucleotides 5′ AATTCGAGATCTGCCGCCATGGTATTGCTGCCTGGGTC 3′(SEQ ID NO. 41) and 5′ TGAGGGTCATGGGCTGGTGG 3′ (SEQ ID NO. 42) usingpGaLV-I1 (Eglitis et al., J. Virol., 67:5472-5477 (1993)) as thetemplate. The 180 base pair PCR product was cut with Bgl11 and Afl11 andthe resulting 110 base pair fragment was isolated. This fragment wasligated in a four-part ligation with a 1.95 kb Afl11-BstE11 fragmentfrom pGaLV-I1, a 4.2 kb Nhe1-Bgl11 fragment from pIK6.1amenvATGUTΔ and aDNA fragment composed of synthetic oligonucleotides 5′-GTAACCTTTAAG 3′(SEQ ID NO. 43) and 5′-CTAGCTTAAAG-3′ (SEQ ID NO. 44) to give pIK6.1GALVSEenv.

pIK6.1MCVGALVgagpol contains the genes encoding the gag and pol proteinsof GALV. It is constructed by replacing the 1.98 kb Bgl11-Nhe MMLVamphotropic envelope encoding fragment of pIK6.1MCVamenvATGUTΔ with thegag/pol sequences from GALV. It is constructed as follows. pGaLV-I1 isfirst digested with Tsp509-1, and then ligated to a DNA fragmentcomposed of synthetic oligonucleotides 5′-GATCTGCCGCCGCCATGGGACAAGAT 3′(SEQ ID NO. 45) and 5′-AATTATCTTGTCCCATGGCGGCGGCA-3′ (SEQ ID NO. 46).This ligation mixture is digested with Rsr11 and the resulting 475 basepair fragment is isolated. pGaLV-I1 is also digested with Afl11 and thenligated to a DNA fragment composed of synthetic oligonucleotides 5′TTAAGCTGCGTATTCGGCGGCGGCGGGACGAGTCTGCAAAATAAG 3′(SEQ ID NO. 47) and 5′CTAGCTTATTTTGCAGACTCGTCCCGCCGCCGCCGAATACGCAGC 3′ (SEQ ID NO. 48). Thisligation mixture is digested with Rsr11 and the resulting 4.59 kbfragment is isolated. These 475 base pair and 4.59 kb fragments,described above, are then ligated in a three-part ligation with a 4.2 kbNhe1-Bgl11 fragment from pIK6.1MCVgagpolATG to producepIK6.1MCVGaLVgagpol.

1. A retroviral packaging plasmid for the production of high titers ofrecombinant retrovirus in human cells comprising one retroviral helperDNA sequence derived from a replication-incompetent retroviral genomeencoding in trans all virion proteins required for packaging areplication-incompetent retroviral vector and for producing virionproteins capable of packaging said replication-incompetent retroviralvector at high titer, without the production of replication-competenthelper virus, said retroviral helper DNA sequence lacking the regionencoding the native enhancer and/or promoter of the viral 5′LTR of saidvirus and lacking both the psi function sequence responsible forpackaging helper genome and the 3′LTR, and encoding a foreign enhancerand/or promoter functional in a selected mammalian cell, and a foreignpolyadenylation site, wherein said helper DNA sequence codes forecotropic MMLV gag and pol and an envelope protein selected from axenotropic, amphotropic, ecotropic or polytropic envelope protein.